WO2019023291A2

WO2019023291A2 - Compositions and methods for making and decoding paired-guide rna libraries and uses thereof

Info

Publication number: WO2019023291A2
Application number: PCT/US2018/043588
Authority: WO
Inventors: Xiaole LIU; Myles Brown; Jingyu PENG; Tengfei XIAO; Wei Li
Original assignee: Dana-Farber Cancer Institute, Inc.
Priority date: 2017-07-25
Filing date: 2018-07-25
Publication date: 2019-01-31
Also published as: WO2019023291A3

Abstract

The present disclosure relates to compositions and methods for making and decoding paired-guide RNA (pgRNA) libraries using the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) system, and using the resulting pgRNA/CRISPR libraries to identify genetic interactions or functional non-coding elements.

Description

COMPOSITIONS AND METHODS FOR MAKING AND DECODING PAIRED-GUIDE

RNA LIBRARIES AND USES THEREOF

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S.

Provisional Application No: 62/536,870, filed July 25, 2017, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates to compositions and methods for making and decoding paired- guide RNA (pgRNA) libraries using the Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) system, and using the pgRNA/CRISPR libraries to identify synthetic lethal genetic interactions (SLGIs) and functional cis-elements (e.g., enhancers).

BACKGROUND OF THE DISCLOSURE

Cancer is a disease in which abnormal cells divide without control and can invade nearby tissues (i.e., metastasize). According to the World Health Organization, cancer is one of the leading causes of morbidity and mortality worldwide, and was responsible for 8.8 million deaths in 2015. Globally, cancer is responsible for nearly 1 in 6 deaths. In 2015, the most common cancer deaths occurred from the following types of cancer: lung cancer (1.69 million deaths), liver cancer (788,000 deaths), colorectal cancer (774,000 deaths), stomach cancer (754,000 deaths), and breast cancer (571,000 deaths).

Cancer is typically treated by any of a variety of methods such as surgery, chemotherapy, radiation therapy, immunotherapy, etc. Unfortunately, many of these methods have

toxic/undesirable side effects. For example, standard chemotherapies for cancer were initially developed based on their ability to kill rapidly dividing cells, and many of their common side effects (e.g., immunosuppression, nausea, hair loss, and the like) are due to their toxic effects on normal tissues that include cell types that undergo rapid division. A central goal of cancer research over the past two decades has been to identify new therapies having great efficaciousness and fewer side effects. To this end, cancer research has focused on discovering tumor-specific traits that may be exploited for selective targeting.

One such approach involves screening for synthetic lethal genetic interactions (SLGIs), which occur when inhibition of two non-essential genes results in a lethal phenotype. The presence of a mutation that inhibits one of the non-essential genes in cancer cells, but not in normal cells, therefore creates an opportunity to selectively kill the cancer cells with a targeted therapy that reduces or eliminates expression of the second non-essential gene. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a revolutionary approach for genome editing and functional genomics research in mammalian systems that may be used to knockout (KO) any pair of genes separately or simultaneously to identify SLGIs, and/or non-coding elements that is essential for cancer cell growth. The development of lentiviral delivery of a genome-scale CRISPR/Cas9 KO library targeting all genes enables both negative and positive selection screening on mammalian cell lines in a cost-effective manner.

Unfortunately, prior art paired-guide RNA (pgRNA) CRISPR/Cas9 KO libraries suffer from the significant disadvantage that they are prone to recombination during construction that creates undesirable constructs, and such libraries are therefore not amenable to scaling. Accordingly, there remains an urgent unmet need for the construction of high-quality, recombination-free pgRNA/CRISPR libraries that allow for reliable, scalable functional genomics studies to identify SLGIs and non-coding elements that may be useful in the treatment of cancer.

SUMMARY OF THE DISCLOSURE

The present disclosure provides paired-guide RNA (pgRNA)/Clustered Regularly- Interspaced Short Palindromic Repeats (CRISPR) libraries having reduced or eliminated rates of internal pgRNA swapping/recombination that may be constructed by using vectors that include two guide RNA (gRNA) cassettes, each having a general structure of promoter-gRNA-scaffold that are constructed from a synthesized oligonucleotide having a general structure of gRNA-1 cassette— unique linker— gRNA-2 cassette such that the unique linker is removed from the final vector containing the two gRNA cassettes. The promoter used in each gRNA cassette may be different, for example, a gRNA-1 cassette may use a human U6 promoter while a paired gRNA-2 cassette may use a mouse U6 promoter. Additionally, the scaffold sequence in each gRNA cassette will typically be different. The present disclosure provides compositions and methods for making and decoding pgRNA libraries using the CRISPR system. Advantageously, the pgRNA/CRISPR libraries disclosed herein may be used to identify synthetic lethal genetic interactions (SLGI) and functional non-coding elements. The techniques provided herein are important because identifying and characterizing SLGIs that occur in combination with tumor suppressor genes may provide novel therapies with which to treat cancer.

In one aspect, the present disclosure provides a paired-guide ribonucleic acid (pgRNA) vector that includes a first guide RNA (gRNA) cassette, a second gRNA cassette; and a

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) expression cassette in which the second gRNA cassette is positioned between the first gRNA cassette and the Cas9 expression cassette.

In one aspect, the disclosure provides an intermediate paired-guide RNA (pgRNA) nucleic acid that includes a first guide RNA (gRNA); a unique linker; and a second gRNA configured so that the unique linker is positioned between the first gRNA and the second gRNA.

In an exemplary embodiment, the first gRNA cassette may include a first nucleic acid sequence including, in 5' to 3' order, a first gRNA promoter, a first gRNA, and a first gRNA scaffold, and the second gRNA cassette may include a second nucleic acid sequence including, in 5' to 3' order, a second gRNA promoter, a second gRNA, and a second gRNA scaffold.

In an exemplary embodiment, the first gRNA promoter may be selected from a mouse U6 promoter, a human U6 promoter, a modified bovine U6 promoter, a mouse HI promoter, a human HI promoter, a mouse 7SK promoter, and a human 7SK promoter, and/or a modified bovine 7SK promoter.

In an exemplary embodiment, the second gRNA promoter may be selected from the group consisting of a mouse U6 promoter, a human U6 promoter, a modified bovine U6 promoter, a mouse HI promoter, a human HI promoter, a mouse 7SK promoter, and a human 7SK promoter, and/or a modified bovine 7SK promoter.

In an exemplary embodiment, the second gRNA promoter may be different than the first gRNA promoter.

In an exemplary embodiment, the first gRNA and the second gRNA may each be between about 17 and 27 nucleotides in length. In an exemplary embodiment, the first gRNA and the second gRNA are each about 19 nucleotides in length. In an exemplary embodiment, the pgRNA vector may be constructed by using an intermediate pgRNA nucleic acid that includes a first gRNA cassette, a unique linker, and a second gRNA cassette in which the unique linker is positioned between the first gRNA cassette and the second gRNA cassette.

In an exemplary embodiment, the unique linker may be between about 10 and 30 nucleotides in length. In an exemplary embodiment, the unique linker may be about 16 nucleotides in length.

In an exemplary embodiment, the Cas9 cassette may include a promoter, a Cas9 coding sequence, and a P2A sequence. In an exemplary embodiment, the promoter may be an EF-l or a CMV promoter.

In an exemplary embodiment the unique linker may have a GC content of less than or equal to 40%.

In one aspect, the present disclosure provides a method of making a paired-guide RNA (pgRNA) library vector that may include the steps of: obtaining a first nucleic acid sequence including, in 5' to 3' order, a first guide RNA (gRNA) cassette promoter, a vector linker, and a second gRNA cassette scaffold; removing the vector linker to create a double strand break (DSB) between a 3' end of the first gRNA cassette promoter and a 5' end of the second gRNA cassette scaffold; inserting into the DSB a second nucleic acid sequence including, in 5' to 3' order, a first guide RNA (gRNA) sequence, a unique linker, and a second gRNA sequence to create an intermediate nucleic acid sequence; removing the unique linker to create a DSB in the intermediate nucleic acid sequence between a 3' end of the first gRNA sequence and a 5' end of the second gRNA sequence; and inserting into the DSB in the intermediate nucleic acid sequence a third nucleic acid sequence including, in 5' to 3' order, a first gRNA cassette scaffold, a spacer, and a second guide RNA (gRNA) cassette promoter, thereby creating the pgRNA vector.

In an exemplary embodiment, the first gRNA cassette promoter may be selected from a mouse U6 promoter and/or a human U6 promoter. In an exemplary embodiment, the second gRNA cassette promoter may be selected from the group consisting of a mouse U6 promoter and/or a human U6 promoter. In an exemplary embodiment, the second gRNA cassette promoter may be different than the first gRNA cassette promoter.

In an exemplary embodiment, the first gRNA sequence and the second gRNA sequence may each be between about 17 and 27 nucleotides in length. In an exemplary embodiment, the first gRNA sequence and the second gRNA sequence may each be about 19 nucleotides in length.

In an exemplary embodiment, the unique linker may be between about 12 and 24 nucleotides in length. In an exemplary embodiment, the unique linker may be about 16 nucleotides in length.

In an exemplary embodiment, the first nucleic acid sequence further includes a Cas9 cassette. In an exemplary embodiment, the Cas9 cassette includes a promoter, a Cas9 coding sequence, and a P2A sequence.

In one aspect, the present disclosure provides a paired-guide RNA (pgRNA)/Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) library that includes: a plurality of pgRNA sequence pairs capable of targeting a plurality of target sequence pairs in a target genome via a CRISPR/Cas9 system to knockout function of a first target sequence and a second target sequence in the target sequence pair, and where the pgRNA vector is constructed by using an intermediate pgRNA nucleic acid, that includes a first guide RNA (gRNA) cassette; a unique linker; and a second gRNA cassette; wherein the unique linker is positioned between the first gRNA cassette and the second gRNA cassette.

In an exemplary embodiment, each of the plurality of pgRNA sequence pairs may include a first guide RNA (gRNA) cassette and a second gRNA cassette.

In an exemplary embodiment, the first gRNA cassette may include a first nucleic acid sequence including, in 5' to 3' order, a first gRNA promoter, a first gRNA sequence, and a first gRNA scaffold, and the second gRNA cassette includes a second nucleic acid sequence including, in 5' to 3' order, a second gRNA promoter, a second gRNA sequence, and a second gRNA scaffold.

In an exemplary embodiment, the first gRNA promoter may be selected from a mouse U6 promoter and/or a human U6 promoter. In an exemplary embodiment, the second gRNA promoter may be selected from a mouse U6 promoter and/or a human U6 promoter. In an exemplary embodiment, the second gRNA promoter may be different than the first gRNA promoter.

In an exemplary embodiment, the unique linker is between about 12 and 24 nucleotides in length. In an exemplary embodiment, the unique linker may be about 16 nucleotides in length.

In one aspect, the present disclosure provides a method of identifying synthetic lethal genetic interaction (SLGI) within a genome that includes the steps of: contacting a population of cells with one or more of the above-described pgRNA vectors; selecting successfully transduced cells; culturing the population of cells for a plurality of population doubling times, wherein genomic DNA may be harvested on a first day of culture and on a last day of culture; deep sequencing the genomic DNA harvested on the first day of culture and on the last day of culture; quantifying abundance of a first guide RNA (gRNA) included in the first gRNA cassette and a second guide RNA (gRNA) included in the second gRNA cassette at the first day of culture and the last day of culture; analyzing an abundance fold change of the first gRNA and the second gRNA between the first day of culture and the last day of culture; and identifying, based on the abundance fold change; a SLGI.

In an exemplary embodiment, the analyzing step further includes a regression residual analysis. In an exemplary embodiment, the analyzing step further includes a BLISS

independence model analysis.

In an exemplary embodiment, the plurality of population doubling times may be between about 8 and 16. In an exemplary embodiment, the plurality of population doubling times may be about 12.

In one aspect, the disclosure provides a tangible, non-transitory, computer-readable media having software encoded thereon, the software, when executed by a processor on a particular device, may be operable to: identify a plurality of gene pairs; determine a response variable; analyze, by a feature selection and regression model, the plurality of gene pairs; and determine, based on the response variable and the analysis, that one or more gene pairs within the plurality of gene pairs interact genetically.

Definitions

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

As used herein, the term "primer" and its derivatives refers generally to any

polynucleotide that can hybridize or anneal to a target sequence of interest. In some

embodiments, the primer can also serve to prime nucleic acid synthesis. Typically, the primer functions as a substrate onto which nucleotides can be polymerized by a polymerase; in some embodiments, however, the primer can become incorporated into the synthesized nucleic acid strand and provide a site to which another primer can hybridize to prime synthesis of a new strand that is complementary to the synthesized nucleic acid molecule. The primer may be comprised of any combination of nucleotides or analogs thereof, which may be optionally linked to form a linear polymer of any suitable length. In some embodiments, the primer is a single- stranded oligonucleotide or polynucleotide. (For purposes of this disclosure, the terms

"polynucleotide" and "oligonucleotide" and "oligo" are used interchangeably herein). In some embodiments, the primer is single-stranded but it can also be double-stranded. The primer optionally occurs naturally, as in a purified restriction digest, or can be produced synthetically. In some embodiments, the primer acts as a point of initiation for amplification or synthesis when exposed to amplification or synthesis conditions; such amplification or synthesis can occur in a template-dependent fashion and optionally results in formation of a primer extension product that is complementary to at least a portion of the target sequence. Exemplary amplification or synthesis conditions can include contacting the primer with a polynucleotide template (e.g., a template including a target sequence), nucleotides and an inducing agent such as a polymerase at a suitable temperature and pH to induce polymerization of nucleotides onto an end of the target- specific primer. If double-stranded, the primer can optionally be treated to separate its strands before being used to prepare primer extension products. In some embodiments, the primer is an oligodeoxyribonucleotide or an oligoribonucleotide. In some embodiments, the primer can include one or more nucleotide analogs. The exact length and/or composition, including sequence, of the target-specific primer can influence many properties, including melting temperature (Tm), GC content, formation of secondary structures, repeat nucleotide motifs, length of predicted primer extension products, extent of coverage across a nucleic acid molecule of interest, number of primers present in a single amplification or synthesis reaction, presence of nucleotide analogs or modified nucleotides within the primers, and the like. In some embodiments, a primer can be paired with a compatible primer within an amplification or synthesis reaction to form a primer pair consisting of a forward primer and a reverse primer. In some embodiments, the forward primer of the primer pair includes a sequence that is

substantially complementary to at least a portion of a strand of a nucleic acid molecule, and the reverse primer of the primer of the primer pair includes a sequence that is substantially identical to at least of portion of the strand. In some embodiments, the forward primer and the reverse primer are capable of hybridizing to opposite strands of a nucleic acid duplex. Optionally, the forward primer primes synthesis of a first nucleic acid strand, and the reverse primer primes synthesis of a second nucleic acid strand, wherein the first and second strands are substantially complementary to each other, or can hybridize to form a double-stranded nucleic acid molecule. In some embodiments, one end of an amplification or synthesis product is defined by the forward primer and the other end of the amplification or synthesis product is defined by the reverse primer. In some embodiments, where the amplification or synthesis of long primer extension products is required, such as amplifying an exon, coding region, or gene, several primer pairs can be created than span the desired length to enable sufficient amplification of the region. In some embodiments, a primer can include one or more cleavable groups. In some embodiments, primer lengths are in the range of about 10 to about 60 nucleotides, about 12 to about 50 nucleotides and about 15 to about 40 nucleotides in length. Typically, a primer is capable of hybridizing to a corresponding target sequence and undergoing primer extension when exposed to amplification conditions in the presence of dNTPs and a polymerase. In some instances, the particular nucleotide sequence or a portion of the primer is known at the outset of the amplification reaction or can be determined by one or more of the methods disclosed herein. In some embodiments, the primer includes one or more cleavable groups at one or more locations within the primer.

As used herein, "polymerase" and its derivatives, generally refers to any enzyme that can catalyze the polymerization of nucleotides (including analogs thereof) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent fashion. Such polymerases can include without limitation naturally occurring polymerases and any subunits and truncations thereof, mutant polymerases, variant polymerases, recombinant, fusion or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof that retain the ability to catalyze such polymerization. Optionally, the polymerase can be a mutant polymerase comprising one or more mutations involving the replacement of one or more amino acids with other amino acids, the insertion or deletion of one or more amino acids from the polymerase, or the linkage of parts of two or more polymerases. Typically, the polymerase comprises one or more active sites at which nucleotide binding and/or catalysis of nucleotide polymerization can occur. Some exemplary polymerases include without limitation DNA polymerases and RNA polymerases. The term "polymerase" and its variants, as used herein, also refers to fusion proteins comprising at least two portions linked to each other, where the first portion comprises a peptide that can catalyze the polymerization of nucleotides into a nucleic acid strand and is linked to a second portion that comprises a second polypeptide. In some embodiments, the second polypeptide can include a reporter enzyme or a processivity-enhancing domain.

Optionally, the polymerase can possess 5' exonuclease activity or terminal transferase activity. In some embodiments, the polymerase can be optionally reactivated, for example through the use of heat, chemicals or re-addition of new amounts of polymerase into a reaction mixture. In some embodiments, the polymerase can include a hot-start polymerase or an aptamer based

polymerase that optionally can be reactivated.

As used herein, "primer/probe set" refers to a grouping of a pair of oligonucleotide primers and an oligonucleotide probe that hybridize to a specific nucleotide sequence. The oligonucleotide set in certain embodiments may include: (a) a forward discriminatory primer that hybridizes to a first location of a nucleic acid sequence or adjacent a particular mutation portion; (b) a reverse discriminatory primer that hybridizes to a second location of the nucleic acid sequence downstream of the first location and (c) preferably a fluorescent probe labeled with a fluorophore and a quencher, which hybridizes to a location of the nucleic acid sequence between the primers. In other words, an oligonucleotide set in certain embodiments consists of a set of specific PCR primers capable of initiating synthesis of an amplicon specific to screening for synthetic lethal genetic interactions (SLGIs) such as, for example, indel or point mutations, and may also include a fluorescent probe that hybridizes to the amplicon. The set may also include in other embodiments a probe with binds to or reacts with one or both of the primers where each or at least one of the primers is modified to contain a marker moiety (e.g., ligand that can be detected with a labeled antibody). As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporated by reference, which describe a method for increasing the concentration of a segment of a polynucleotide of interest in a mixture of genomic DNA without cloning or purification. This process for amplifying the polynucleotide of interest consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired polynucleotide of interest, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded polynucleotide of interest. To effect amplification, the mixture is denatured and the primers then annealed to their

complementary sequences within the polynucleotide of interest molecule. Following annealing, the primers are extended with a polymerase to form a new pair of complementary strands. The steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle"; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired polynucleotide of interest. The length of the amplified segment of the desired polynucleotide of interest (amplicon) is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of repeating the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the polynucleotide of interest become the predominant nucleic acid sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified." As defined herein, target nucleic acid molecules within a sample including a plurality of target nucleic acid molecules are amplified via PCR. In a modification to the method discussed above, the target nucleic acid molecules can be PCR amplified using a plurality of different primer pairs, in some cases, one or more primer pairs per target nucleic acid molecule of interest, thereby forming a multiplex PCR reaction. Using multiplex PCR, it is possible to simultaneously amplify multiple nucleic acid molecules of interest from a sample to form amplified target sequences. It is also possible to detect the amplified target sequences by several different methodologies (e.g., quantitation with a bioanalyzer or qPCR, hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P- labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified target sequence). Any oligonucleotide sequence can be amplified with the appropriate set of primers, thereby allowing for the amplification of target nucleic acid molecules from genomic DNA, cDNA, formalin-fixed paraffin-embedded DNA, fine-needle biopsies and various other sources. In particular, the amplified target sequences created by the multiplex PCR process as disclosed herein, are themselves efficient substrates for subsequent PCR amplification or various downstream assays or manipulations.

The methods disclosed herein also contemplate any other type of amplification reaction or modified PCR reaction known in the art, which may include, but are not limited to: Allele- specific PCR; Assembly PCR or Polymerase Cycling Assembly (PCA); Digital PCR (dPCR); Helicase-dependent amplification; Hot start PCR; In silico PCR; Intersequence-specific PCR (ISSR); Inverse PCR; Ligati on-mediated PCR; Methylati on-specific PCR (MSP); Miniprimer PCR; Multiplex Ligation-dependent Probe Amplification (MLPA); Multiplex-PCR;

Nanoparticle-Assisted PCR (nanoPCR); Nested PCR; Overlap-extension PCR or Splicing by overlap extension (SOEing); PAN-AC (uses isothermal conditions for amplification and may be used in living cells); Quantitative PCR (qPCR); Reverse Transcription PCR (RT-PCR); Solid Phase PCR; Suicide PCR; Thermal asymmetric interlaced PCR (TAIL-PCR); Touchdown PCR (Step-down PCR); Universal Fast Walking; and the like.

As defined herein, the term "sample" and its derivatives, is used in its broadest sense and includes any specimen, culture and the like that is suspected of including a target. In some embodiments, the sample comprises DNA, RNA, PNA, LNA, chimeric, hybrid, or multiplex- forms of nucleic acids. The sample can include any biological, clinical, surgical, agricultural, atmospheric or aquatic-based specimen containing one or more nucleic acids. The term also includes any isolated nucleic acid sample such as genomic DNA, fresh-frozen or formalin-fixed paraffin-embedded nucleic acid specimen, and the like.

As used herein, "patient" or "subject" can mean either a human or non-human animal, preferably a mammal having a tumor, cancer, or otherwise a proliferative disorder. By "subject" is meant any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds. A human subject may be referred to as a patient. It should be noted that clinical observations described herein were made with human subjects and, in at least some embodiments, the subjects are human.

As used herein, "kits" are understood to contain at least one non-standard laboratory reagent for use in the methods of the disclosure in appropriate packaging, optionally containing instructions for use. The kit can further include any other components required to practice the method of the disclosure, as dry powders, concentrated solutions, or ready to use solutions. In some embodiments, the kit comprises one or more containers that contain reagents for use in the methods of the disclosure; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding reagents.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

Where applicable or not specifically disclaimed, any one of the embodiments described herein are contemplated to be able to combine with any other one or more embodiments, even though the embodiments are described under different aspects of the disclosure.

These and other embodiments are disclosed and/or encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but not intended to limit the disclosure solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which:

FIG. 1 depicts a paired-guide (pgRNA) library oligonucleotide design and the swapping pair issues that are generated from polymerase chain reaction (PCR). This design includes an oligonucleotide pool that contains a common linker between two guide RNA (gRNA) sequences. During each round of PCR, the non-full length PCR products base pair with each other through the common linker, which serves as an anchor. The 3'->5' exonuclease activity of the polymerase may digest the unmatched gRNA sequence when two ssDNAs bind to each other through the common linker. After the extension step, recombination may occur between different gRNA pairs, leading to the creation of undesired gRNA pairs.

FIGS. 2A-2F depict the results of two rounds of CRISPR screens on T47D and MCF7 cell lines that revealed that ER-regulated C-Src Tyrosine Kinase (CSK) mediates hormone independent breast cancer cell growth and is synthetic lethal in combination with P21 (RACl) Activated Kinase 2 (PAK2). FIG. 2A is a schematic that shows the experimental procedure for the first round of CRISPR screening. FIG. 2B is a graph that shows that CSK is positively selected in both T47D and MCF7 cells cultured in hormone depleted medium treated with vehicle conditions compared to Estradiol (E2). FIG. 2C is a graph that shows the frequency change of the CSK-targeting single-guide RNAs (sgRNAs) in both screens. FIG. 2D is a plate staining assay that depicts the effects on cell growth by knocking out CSK using three different gRNAs against CSK, and one gRNA against AAVS1 as a control. CSK function is rescued by the expression of gRNA-resistant CSK cDNAs in these CSK null cells. Cell growth was measured by crystal violet staining assays. FIG. 2E is a schematic that shows the experimental procedures of the second round of CRISPR screening in which T47D cells were first infected with lentiviral gCSK and gAAVSl . After blasticidin selection, T47D cells were generated with stable expression of gCSK and gAAVSl, respectively, and then the genome-wide CRISPR screens were performed in the same manner as the first round. FIG. 2F depicts a Western blot and bar graphs that validate the presence of a synthetic lethal interaction between PAK2 and CSK in T47D cells.

FIGS. 3A-3I depict the pgRNA CRISPR library construction and screening strategy according to an exemplary embodiment of the disclosure. FIG. 3 A is a flowchart that depicts a two-step pgRNA cloning strategy. Briefly, a synthesized DNA oligo including the sequences of two gRNAs (represented in red and purple) with an identical linker (grey, in contrast to the unique linkers in the improved oligo design described herein to avoid swapping) was amplified using primers targeting flanking sequence to generate a double-stranded DNA molecule containing 40-80 bp homologies to the U6 promoter and the gRNA scaffold. A Gibson assembly reaction was performed between the amplified fragments and the BsmBI digested gRNA- expressing backbone, and then transformed into competent bacterial cells. This intermediate construct was then digested by BsmBI and a ligation was performed with BsmBI digested tracrRNA-linker-U6 segment. FIG. 3B shows DNA sequences of the engineered oligo and linker between the two gRNAs of each pair (SEQ ID NO: 29). FIG. 3C shows a schematic of pgRNA cell library construction and screening procedures in which the pgRNA library was delivered into a Cas9-expressing cell line of interest by lentiviral infection with a MOI of about 0.3, and the infected cells were harvested by FACS for green fluorescence 3 days' post-infection. For screening, library cells were cultured for 30 days before genome DNA extraction and high- throughput sequencing analysis of the barcode gRNA regions. FIG. 3D shows an improved pgRNA vector including two gRNA cassettes and a Cas9 expression cassette according to an exemplary embodiment. FIG. 3E shows a method of making the improved pgRNA vector of FIG. 3D. FIG. 3F shows the design of the synthesized oligonucleotide including a first gRNA, a unique linker flanked by to restriction sites, and a second gRNA (SEQ ID NO: 16). FIG. 3G is a schematic showing how the method of FIG. 3E reduces frequencies of recombination/swapping of pgRNAs during library construction. FIG. 3H shows two graphs depicting the read count distribution of correct pgRNAs and swapped/recombined pgRNAs on the pgRNA plasmid library and the read count distribution on Day 0, Cell lthe cell library. FIG. 31 shows the table of colony PCR amplicons and sequencing analysis result.

FIG. 4 depicts a graph showing an exemplary regression residual approach to identify SLGI from a pgRNA screen. The Y-axis represents the logFC of pgRNA targeting a pair of TSG with partner, whereas the X-axis represents the logFC of pgRNA targeting a pair of AAVSl with the same partner. Ideally, each SLGI of a gene should be supported by multiple pgRNAs. Under certain circumstances, synthetic rescue effect might be observed.

FIGS. 5A-D generally depict library design and gene calling for exemplary CRISPR screens. FIG. 5 A is a schematic that shows a sequence logo illustrating the features that contribute to sgRNA efficiency. FIG. 5B includes a gel and a bar graph that shows that indel rates of the sgRNAs are predicted to be inefficient (predicted low) or efficient (predicted high). FIG. 5C is a table that shows an example design matrix of MAGeCK-MLE according to an exemplary embodiment of the disclosure in which 1 indicates the presence of a certain treatment such as, for example, adding a drug or chemical compound, removing a growth factor, etc., in a sample. FIG. 5D is a schematic that shows the initialization and iterative update of the EM model according to the MAGeCK algorithm. FIG. 6 is a graph that depicts performance of a prediction algorithm with feature selection and a regression residual approach according to the techniques herein. The model was trained on known yeast SLGI pairs and TCGA colon cancer data, and tested on human SLGI pairs from a shRNA screen on HTC116 colon cancer cells. Using the 1204 identified GI pairs as true positives and randomly selected 1000 non-GI pairs as true negatives, the algorithm provides a clear separation of the two (p-value < 2.2e-16).

FIG. 7 is an equation that represents a weighted regression to combine different training datasets for SLGI prediction. For each data set, a weight score may be derived from cross- validation with a R2 metric, where R2 is the coefficient of determination (R^A2) in regression. The final coefficient for each SLGI features may be solved through weighted least square method.

FIGS. 8A-C depict generally the characterization of the mechanisms of pan-cancer or cancer-specific SLGIs. FIG. 8A depicts a schematic demonstrating pan-cancer and cancer- specific SLGIs. FIG. 8B is a schematic that shows putative effects of pan-cancer SLGI on downstream gene expression. FIG. 8C is a schematic that shows putative effects of cancer- specific SLGI on cell number and downstream gene expression. In scenario 1, a downstream pathway is regulated similarly between different cancers but differentially required. In scenario 2, a downstream pathway is expressed differentially between cancers, which can be attributable to different expression of regulators.

FIGS. 9A-9B depict schematic overview of using an exemplary pgRNA library of the disclosure to conduct a functional enhancer screen (FIG. 9A) and a schematic of the screening protocol (FIG. 9B).

FIG. 10 shows six two schematics and two graphs providing data about the deletion of a CSK enhancer according to an exemplary embodiment of the disclosure. The upper portion of FIG. 10 presents a schematic that shows the location of one CSK enhancer (left schematic) and a schematic that shows the designed gRNA targeting loci around this enhancer (right schematic). The bottom portion of FIG. 10 shows CSK expression levels upon introduction of different pairs of gRNAs with indicated time of estrogen treatment (0, 1, 4 hours) in T47D (left graph) and MCF7 (right graph) cell lines. FIG. 11 shows a schematic of the CSK enhancer tilling design in which more than 1,300 pgRNAs (black stick pairs in the second row) were designed in a tilling format to cover the CSK enhancer region with indicated DNasel-, ER-, FoxAl-, GAT A3 - binding peaks.

FIG. 12 shows a schematic, a table, and a dot plot describing the analysis of the CSK enhancer tilling according to an exemplary embodiment of the disclosure. The top schematic shows the use of bins to convert overlapping pgRNA target regions into consecutive units on genomic DNA. The bottom left table shows the exemplary relationship between pgRNAs and bins, and the use of bins as genes to run MAGeCK to evaluate the change of each bin, while the bottom right dot plot is the MAGeCK result, showing the p-value distribution of the positively- selected bins.

FIG. 13 shows a schematic of a region with > 1,300 pgRNAs and a similar schematic associated with dot plots of data derived from positive and negative selection experiments. The left schematic shows the location of the pgRNA-tilling covered enhancer region and CSK expression cassette, along with indicated DNasel, ESR1-, FoxAl-, GATA3- and H3K27ac peaks. The right schematic shows the screening results indicating that both the known enhancer (the right arrow) and potential novel enhancers (the left two arrows) were identified.

FIG. 14 is a chart showing the pgRNA selection matrix. Out of a total of 49 possible pairwise gRNA combinations for a given gene pair, each gene has 7 unique CRISPR gRNAs. The indicated 21 combinations are chosen to ensure that each gRNA is used three times.

FIG. 15 is a chart showing quality control of the 15K pgRNA library. Quality control was assessed for both plasmid and cell libraries by paired-end pgRNA sequencing to ensure the coverage and evenness of all designed pgRNAs and to check for swapping/recombination events.

FIG. 16 is a chart showing the MAGeCK/RRA analysis result of the functional positive control SLGI pairs in the CRISPR screen.

FIG. 17A-FIG. 17D are a series of dot plots showing the analysis of the 15K pgRNA library screen. FIG. 17A is a dot plot anchored on RBI . FIG. 17B is a dot plot anchored on PEN. FIG. 17C is a dot plot anchored on NF1. FIG. 17D is a dot plot anchored on CSK.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure is based, at least in part, on the discovery that paired-guide RNA (pgRNA)/Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) libraries having reduced or eliminated rates of internal pgRNA swapping/recombination that may be constructed by using vectors that include two guide RNA (gRNA) cassettes, each having a general structure of promoter-gRNA-scaffold that are constructed from a synthesized oligonucleotide having a general structure of gRNA-1 cassette— unique linker— gRNA-2 cassette such that the unique linker is removed from the final vector containing the two gRNA cassettes.

The promoter used in each gRNA cassette may be different, for example, a gRNA-1 cassette may use a human U6 promoter while a paired gRNA-2 cassette may use a mouse U6 promoter. Additionally, the scaffold sequence in each gRNA cassette will typically include a trans-activating crRNA (tracrRNA), which may include sequences in addition to the tracrRNA. Exemplary human and mouse U6 promoter sequences and RNA scaffolds sequences are listed as below:

1. human U6 promoter (SEQ ID NO: 11):

GAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAG TAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATA TGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGG ACGAAACACCG

2. mouse U6 promoter (SEQ ID NO: 12):

GATCCGACGCGCCATCTCTAGGCCCGCGCCGGCCCCCTCGCACGGACTTGTGGGAG

AAGCTCGGCTACTCCCCTGCCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATA

GAGGCTTAATGTGCGATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATT

TTACATGATAGGCTTGGATTTCTATAACTTCGTATAGCATACATTATACGAAGTTATA

AACAGCACAAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTAT

AAGTATCCCTTGGAGAACCACCTTGTTG

3. 1st gRNA scaffold in the hU6 cassette (SEQ ID NO: 13):

GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

4. 2nd gRNA scaffold in the mU6 cassette (SEQ ID NO: 14): GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA AGTGGCACCGAGTCGGTGCTTTTTT

5. An exemplary vector may include (SEQ ID NO: 15):

TATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAAT

TAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAA

AGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCA

TATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAA

GGACGAAACACCGCCTCCCGCTCCTGGAGCGG

[gRNAl in bold]

GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC

AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTCGAGTACTAGGATCCATTA

GGCGGCCGCGTCGACAAGCTTTCTAGAGAATTCGATCCGACGCGCCATCTCTAGGCC

CGCGCCGGCCCCCTCGCACGGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGG

TTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGAC

AGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTAT

AACTTCGTATAGCATACATTATACGAAGTTATAAACAGCACAAAAGGAAACTCACC

CTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAGTATCCCTTGGAGAACCACCT

TGTTGGATATTCACCATTATAGGT

[gRNA2 in bold]

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAA

AGTGGCACCGAGTCGGTGCTTTTTTGAATTCTAGACTTGATGCTAACTAGGTCTTGA

AAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGT.

In an exemplary embodiment, the vectors described herein may include portions of the lentiCRISPRv2 vector (e.g., the World Wide Web at (www) addgene.org/52961/).

The present disclosure provides compositions and methods for making and decoding pgRNA libraries using the CRISPR system. Advantageously, the pgRNA/CRISPR libraries disclosed herein may be used to identify synthetic lethal genetic interactions (SLGI) and non- coding functional elements or cis-elements. The techniques provided herein are important because identifying and characterizing SLGI that occur in combination with cancer causing genes (e.g., tumor suppressor genes) may provide novel therapies with which to treat cancer. In this regard, the techniques herein provide experimental and computational methods for the large- scale identification of novel therapies to treat cancers with tumor suppressor loss.

Overview

Cancer may be driven by the activation of oncogenes or the deactivation of tumor suppressor genes (TSGs). For example, cancer may be cause by gain-of-function mutations in oncogenes and loss-of-function mutations in TSGs. While activating oncogenic mutations may often be targeted directly by therapeutic intervention, successfully restoring the function of a TSG has thus far not been possible in the clinic. While activating oncogenic mutations may often be directly targeted by therapeutic intervention, successful treatment for tumor suppressor loss has thus far been challenging in the clinic.

Genetic interaction is a phenomenon in which the phenotype of mutations in two genes differs significantly from each mutation's individual effects. In extreme cases, genetic interaction may give rise to synthetic lethality when inactivation of two nonessential genes results in a lethal phenotype. Such synthetic lethal genetic interactions (SLGI) may provide insights on novel cancer therapeutic targets or target combinations that may enhance the efficacy and specificity of targeted drugs. Over the past few years, there have been tremendous efforts to identify SLGI genes in the cancer genome with the primary aim of identifying novel therapeutic targets among the synthetic lethal partners of dysfunctional TSGs. Unfortunately, the accuracy and cost effectiveness of prior art techniques for identifying and validating SLGI pairs in mammalian systems is not sufficient to allow identification of SLGIs at scale.

Historically, many genomic technologies have been developed to map SLGIs in model organisms and humans. For example, two projects of genome-wide quantitative mapping of synthetic lethal interactions have been conducted in yeast based on gene deletion strains (see e.g., references 1 and 2). Based on the same technology, another study screened potential interactions among orthologs of human TSGs and genes encoding drug targets in yeast (see e.g., reference 3). SLGI mapping by directed gene disruptions in human cell lines is very important, as SLGIs involving TSGs or oncogenes may provide insights to precision cancer medicine. Such disruptions generally use RNA interference (e.g., siRNA or shRNA) knockdown or

CRISPR/Cas9 knockout and can be roughly categorized as either a "1 x n" design or an "a x b" design. In a "1 x n" design, genome-wide (n genes) RNAi or CRISPR screens may be used to identify genes showing differential essentiality between cell lines where an anchor gene (1 gene) is active vs inactive. Here, the anchor gene may be inactivated by RNAi or CRISPR (see e.g., references 4-6), drug inhibition (see e.g., reference 7), or inherently lost in the cell line (see e.g., reference 8).

In an "a x b" design, all pairwise combinations of shRNAs or CRISPR guide RNAs (gRNAs) within a starting pool may be randomly combined together to test possible interactions among sets of genes. Although "a" and "b" could theoretically be different, so far published screens are mostly in an "a x a" design, such as 190 x 190 shRNA pairs (see e.g., reference 9) or 153 x 153 CRISPR gRNA pairs (see e.g., reference 10). SLGI screens on specific pairs using simultaneous delivery of two shRNAs have been proposed by The DECIPHER project, although to date no studies have been published using this technique and shRNA is unfortunately known to have significant off-target effect. The "a x b" design may also be carried out in arrayed format with automated technologies (see e.g., reference 1 1) instead of pooled screens. However, such combinatorial design falls short of the required throughput to interrogate the potential interaction space of all the possible SLGIs involving TSGs.

Many computational approaches have also been developed to systematically study genetic interactions for yeast where genome-wide experimental maps of SLGIs are available (see e.g., references 12-15). In cancer, SLGI has been computationally predicted through mapping yeast genetic interactions to their human orthologs (see e.g., reference 16) and utilizing metabolic models and evolutionary characteristics of metabolic genes (see e.g., references 17- 19). With the rapidly accumulating cancer genomic data, a data-driven method, named DAISY, was used to integrate somatic copy number alterations, shRNA-based essentiality screens, and co-expression patterns on hundreds of cancer cell lines to detect SLGI pairs in human (see e.g., reference 20). However, the filtering criterion integrating each data type is determined in an ad hoc manner, and the experimental validation was only conducted on a handful of interactions (see e.g., references 20). Despite these efforts, SLGI identification and validation has been limited by the scale and accuracy of the prior art experimental technology; therefore, it is very difficult to systematically evaluate the performance of SLGI computational predictions with prior art methodologies. Despite the success of targeted therapies treating cancers with activating mutations, prior art attempts to therapeutically target cancers with TSGs loss (e.g., Tumor Protein P53 (TP53), Phosphatase and Tensin Homolog (PTEN), and the like) have not been effective. SLGI could provide novel insights on therapeutic targets to treat cancers with TSG loss. There have been tremendous efforts to identify SLGI among genes in the genome; unfortunately, the accuracy and cost effectiveness of these efforts in mammalian systems was not sufficient to allow SLGI screening at scale. The CRISPR/Cas9 genome editing technology and CRISPR/Cas9 knockout (KO) screens offers exciting new opportunities to investigate SLGI in mammalian genomes. CRISPR

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a revolutionary approach for genome editing and functional genomics research in mammalian systems. Cas9 nucleases are directed to specific genomic loci by single-guide RNAs (sgRNAs) containing 19-20 nucleotides that are complementary to the target DNA sequences, thereby creating frameshift insertion/deletion (indel) mutations that result in a loss-of-function allele. The development of lentiviral delivery of a genome-scale CRISPR/Cas9 knockout (KO) library targeting all genes enables both negative and positive selection screening on mammalian cell lines in a cost-effective manner. In genome-wide sgRNA/CRISPR screens, each gene may be targeted by several sgRNAs for KO, and the mutant pool carrying different gene KOs can then be resolved by high throughput sequencing. Those sgRNA targeting genes that inhibit growth under the screening conditions will be enriched while those targeting essential genes will be under-represented. Thus, CRISPR screening is a powerful technology for systematic genetic analysis, and is especially relevant in cancer where growth under various conditions or under drug selection is a critical phenotype.

The delivery of the two sgRNAs into a single cell could create mutations at both targeting locus simultaneously or fragment deletions if two cutting sites are close to each other. Therefore, to build the CRISPR library in which each vector express two gRNAs provides a new approach to investigate gene interactions and functional non-coding elements in a systematic way.

Although sgRNA/CRISPR libraries have been constructed and used for screening, prior art libraries suffer from the significant disadvantage that they are prone to recombination that creates undesirable sgRNA pairs and are therefore not amenable to scaling. Accordingly, there remains an urgent unmet need for the construction of high-quality, recombination-free pgRNA/CRISPR libraries that allow for reliable, scalable functional genomics studies to identify synthetic lethal gene interactions and non-coding elements.

It is contemplated within the scope of the disclosure, that the CRISPR/Cas system may be used to modify any of the nucleotides described herein, either for in vitro or in vivo manipulation of the nucleotides, or for identification of genetic interactions (e.g., SLGIs). For example, the techniques herein provide that the CRISPR/Cas system may be used therapeutically to down regulate expression of, or knockout, pairs of genes in a cancer cell(s). The CRISPR/Cas system is abundantly described in US Patent No. 8,795,965, US Patent No. 8,889,356, US Patent No. 8,771,945, US Patent No. 8,889,418, and US Patent No. 8,895,308, which are hereby

incorporated by reference in their entirety.

Briefly, the term "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a trans-activating CRISPR (tracr) sequence (e.g.

tracrRNA), a tracr-mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (e.g., guide RNA), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system may be derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (e.g., a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, "target sequence" refers to a sequence to which a guide sequence (e.g., gRNA) is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA

polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be within an organelle of a eukaryotic cell, for example, mitochondrion or chloroplast. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a revolutionary approach for genome editing of mammalian systems. Cas9 nucleases are directed to specific genomic loci by single-guide RNAs (sgRNAs) containing 17-27 nucleotides that are complementary to the target DNA sequences and have the ability to create frameshift

insertion/deletion (indel) mutations that result in a loss-of-function allele. In an exemplary embodiment, the sgRNAs may be 19-20 nucleotides in length. In an exemplary embodiment, the sgRNAs may be 19 nucleotides in length. Recently, the development of lentiviral delivery of a genome-scale CRISPR/Cas9 knockout (KO) library targeting all genes enables both negative and positive selection screening on mammalian cell lines in a cost-effective manner (see e.g., references 7, 21, and 22). In genome-wide CRISPR KO screens, each gene is targeted by several sgRNAs for KO, and the mutant pool carrying different gene KOs can then be resolved by high throughput sequencing. Those sgRNA targeting genes that inhibit growth under the screening conditions will be enriched while those targeting essential genes will be under-represented. Thus, CRISPR screening is a powerful technology for systematic genetic analysis, and is especially relevant in cancer where growth under various conditions or under drug selection is a critical phenotype.

Over the last few years, techniques have been developed to provide CRISPR screens using paired guide RNAs (pgRNAs) to create deletions in, or to silence, two different genes simultaneously. For example, studies have found that pgRNAs driven by separate U6 promoters work better than consecutive gRNAs transcribed from the same U6 (see e.g., references 23-25). To prevent the two gRNA expression cassettes (U6-gRNA-tracrRNA) from swapping (e.g., recombining) during lentiviral replication, U6 promoters from different species and different tracr RNA sequences for the two gRNAs may be used (see e.g., references 25). This approach also enables the pgRNAs to be read from paired-end sequencing. Unfortunately, pilot studies have indicated that the pgRNAs may still swap or recombine at two different stages during the pooled screen. First, when the synthesized long oligonucleotides carrying the two gRNAs are PCR amplified to construct the custom CRISPR library, the two gRNAs may swap or recombine during PCR due to the common restriction enzyme recognition sites and linker sequence that are shared between the two gRNAs (see e.g., FIG. 1). Second, during the final PCR step to prepare the sequencing library, the two gRNAs may swap or recombine again during PCR due to the first tracrRNA and second U6 sequences that are shared in common between the two gRNAs. Additionally, the polymerase used in current PCR reactions has a 3' to 5' exonuclease activity that exacerbates the frequency of swapping or recombining during the PCR process (see e.g., FIG. 1). For example, long non-coding RNA (IncRNA) deletion CRISPR screens used 25 pgRNAs to delete the promoter of each IncRNA; however, this deletion screen still suffered from a high false negative rate due to recombination between pgRNAs during PCR (see e.g., reference 23). The techniques herein provide the ability to finally resolve the PCR

swapping/recombination issues inherent in the prior art (see e.g., reference 23) and provide a pooled pgRNA CRISPR screening methodology that is robust, effective, accurate, and scalable.

By "Tumor Protein P53 (TP53) nucleic acid molecule" is meant a polynucleotide encoding a TP53 polypeptide. An exemplary TP53 nucleic acid molecule is provided at NCBI Accession No. NM_000546, version NM_000546.5, incorporated herein by reference, and reproduced below (SEQ ID NO: 1):

1 gatgggattg gggttttccc ctcccatgtg ctcaagactg gcgctaaaag ttttgagctt 61 ctcaaaagtc tagagccacc gtccagggag caggtagctg ctgggctccg gggacacttt 121 gcgttcgggc tgggagcgtg ctttccacga cggtgacacg cttccctgga ttggcagcca 181 gactgccttc cgggtcactg ccatggagga gccgcagtca gatcctagcg tcgagccccc 241 tctgagtcag gaaacatttt cagacctatg gaaactactt cctgaaaaca acgttctgtc 301 ccccttgccg tcccaagcaa tggatgattt gatgctgtcc ccggacgata ttgaacaatg 361 gttcactgaa gacccaggtc cagatgaagc tcccagaatg ccagaggctg ctccccccgt 421 ggcccctgca ccagcagctc ctacaccggc ggcccctgca ccagccccct cctggcccct 481 gtcatcttct gtcccttccc agaaaaccta ccagggcagc tacggtttcc gtctgggctt 541 cttgcattct gggacagcca agtctgtgac ttgcacgtac tcccctgccc tcaacaagat 601 gttttgccaa ctggccaaga cctgccctgt gcagctgtgg gttgattcca cacccccgcc 661 cggcacccgc gtccgcgcca tggccatcta caagcagtca cagcacatga cggaggttgt 721 gaggcgctgc ccccaccatg agcgctgctc agatagcgat ggtctggccc ctcctcagca 781 tcttatccga gtggaaggaa atttgcgtgt ggagtatttg gatgacagaa acacttttcg 841 acatagtgtg gtggtgccct atgagccgcc tgaggttggc tctgactgta ccaccatcca 901 ctacaactac atgtgtaaca gttcctgcat gggcggcatg aaccggaggc ccatcctcac 961 catcatcaca ctggaagact ccagtggtaa tctactggga cggaacagct ttgaggtgcg 1021 tgtttgtgcc tgtcctggga gagaccggcg cacagaggaa gagaatctcc gcaagaaagg 1081 ggagcctcac cacgagctgc ccccagggag cactaagcga gcactgccca acaacaccag 1141 ctcctctccc cagccaaaga agaaaccact ggatggagaa tatttcaccc ttcagatccg 1201 tgggcgtgag cgcttcgaga tgttccgaga gctgaatgag gccttggaac tcaaggatgc 1261 ccaggctggg aaggagccag gggggagcag ggctcactcc agccacctga agtccaaaaa 1321 gggtcagtct acctcccgcc ataaaaaact catgttcaag acagaagggc ctgactcaga 1381 ctgacattct ccacttcttg ttccccactg acagcctccc acccccatct ctccctcccc 1441 tgccattttg ggttttgggt ctttgaaccc ttgcttgcaa taggtgtgcg tcagaagcac 1501 ccaggacttc catttgcttt gtcccggggc tccactgaac aagttggcct gcactggtgt 1561 tttgttgtgg ggaggaggat ggggagtagg acataccagc ttagatttta aggtttttac 1621 tgtgagggat gtttgggaga tgtaagaaat gttcttgcag ttaagggtta gtttacaatc 1681 agccacattc taggtagggg cccacttcac cgtactaacc agggaagctg tccctcactg 1741 ttgaattttc tctaacttca aggcccatat ctgtgaaatg ctggcatttg cacctacctc 1801 acagagtgca ttgtgagggt taatgaaata atgtacatct ggccttgaaa ccacctttta 1861 ttacatgggg tctagaactt gacccccttg agggtgcttg ttccctctcc ctgttggtcg 1921 gtgggttggt agtttctaca gttgggcagc tggttaggta gagggagttg tcaagtctct 1981 gctggcccag ccaaaccctg tctgacaacc tcttggtgaa ccttagtacc taaaaggaaa 2041 tctcacccca tcccacaccc tggaggattt catctcttgt atatgatgat ctggatccac 2101 caagacttgt tttatgctca gggtcaattt cttttttctt tttttttttt ttttttcttt 2161 ttctttgaga ctgggtctcg ctttgttgcc caggctggag tggagtggcg tgatcttggc 2221 ttactgcagc ctttgcctcc ccggctcgag cagtcctgcc tcagcctccg gagtagctgg 2281 gaccacaggt tcatgccacc atggccagcc aacttttgca tgttttgtag agatggggtc 2341 tcacagtgtt gcccaggctg gtctcaaact cctgggctca ggcgatccac ctgtctcagc 2401 ctcccagagt gctgggatta caattgtgag ccaccacgtc cagctggaag ggtcaacatc 2461 ttttacattc tgcaagcaca tctgcatttt caccccaccc ttcccctcct tctccctttt 2521 tatatcccat ttttatatcg atctcttatt ttacaataaa actttgctgc cacctgtgtg 2581 tctgaggggt g

By "Tumor Protein P53 (TP53) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_000537, version NP 000537.3, incorporated herein by reference, as reproduced below (SEQ ID NO: 2):

1 meepqsdpsv epplsqetfs dlwkllpenn vlsplpsqam ddlmlspddi eqwftedpgp

61 deaprmpeaa ppvapapaap tpaapapaps wplsssvpsq ktyqgsygfr lgflhsgtak

121 svtctyspal nkmfcqlakt cpvqlwvdst pppgtrvram aiykqsqhmt e vrrcphhe

181 rcsdsdglap pqhlirvegn lrveylddrn tfrhs vvpy eppevgsdct tihynymcns

241 scmggmnrrp iltiitleds sgnllgrnsf evrvcacpgr drrteeenlr kkgephhelp

301 pgstkralpn ntssspqpkk kpldgeyftl qirgrerfem frelnealel kdaqagkepg

361 gsrahsshlk skkgqstsrh kklmfktegp dsd

By "Phosphatase and Tensin Homolog (PTEN) nucleic acid molecule" is meant a polynucleotide encoding a PTEN polypeptide. An exemplary PTEN nucleic acid molecule is provided at NCBI Accession No. NM_000314, version NM_000314.6, incorporated herein by reference, and reproduced below (SEQ ID NO: 3):

1 cctcccctcg cccggcgcgg tcccgtccgc ctctcgctcg cctcccgcct cccctcggtc

61 ttccgaggcg cccgggctcc cggcgcggcg gcggaggggg cgggcaggcc ggcgggcggt 121 gatgtggcgg gactctttat gcgctgcggc aggatacgcg ctcggcgctg ggacgcgact 181 gcgctcagtt ctctcctctc ggaagctgca gccatgatgg aagtttgaga gttgagccgc 241 tgtgaggcga ggccgggctc aggcgaggga gatgagagac ggcggcggcc gcggcccgga 301 gcccctctca gcgcctgtga gcagccgcgg gggcagcgcc ctcggggagc cggccggcct 361 gcggcggcgg cagcggcggc gtttctcgcc tcctcttcgt cttttctaac cgtgcagcct 421 cttcctcggc ttctcctgaa agggaaggtg gaagccgtgg gctcgggcgg gagccggctg 481 aggcgcggcg gcggcggcgg cacctcccgc tcctggagcg ggggggagaa gcggcggcgg 541 cggcggccgc ggcggctgca gctccaggga gggggtctga gtcgcctgtc accatttcca 601 gggctgggaa cgccggagag ttggtctctc cccttctact gcctccaaca cggcggcggc 661 ggcggcggca catccaggga cccgggccgg ttttaaacct cccgtccgcc gccgccgcac 721 cccccgtggc ccgggctccg gaggccgccg gcggaggcag ccgttcggag gattattcgt 781 cttctcccca ttccgctgcc gccgctgcca ggcctctggc tgctgaggag aagcaggccc 841 agtcgctgca accatccagc agccgccgca gcagccatta cccggctgcg gtccagagcc 901 aagcggcggc agagcgaggg gcatcagcta ccgccaagtc cagagccatt tccatcctgc 961 agaagaagcc ccgccaccag cagcttctgc catctctctc ctcctttttc ttcagccaca 1021 ggctcccaga catgacagcc atcatcaaag agatcgttag cagaaacaaa aggagatatc 1081 aagaggatgg attcgactta gacttgacct atatttatcc aaacattatt gctatgggat 1141 ttcctgcaga aagacttgaa ggcgtataca ggaacaatat tgatgatgta gtaaggtttt 1201 tggattcaaa gcataaaaac cattacaaga tatacaatct ttgtgctgaa agacattatg 1261 acaccgccaa atttaattgc agagttgcac aatatccttt tgaagaccat aacccaccac 1321 agctagaact tatcaaaccc ttttgtgaag atcttgacca atggctaagt gaagatgaca 1381 atcatgttgc agcaattcac tgtaaagctg gaaagggacg aactggtgta atgatatgtg 1441 catatttatt acatcggggc aaatttttaa aggcacaaga ggccctagat ttctatgggg 1501 aagtaaggac cagagacaaa aagggagtaa ctattcccag tcagaggcgc tatgtgtatt 1561 attatagcta cctgttaaag aatcatctgg attatagacc agtggcactg ttgtttcaca 1621 agatgatgtt tgaaactatt ccaatgttca gtggcggaac ttgcaatcct cagtttgtgg 1681 tctgccagct aaaggtgaag atatattcct ccaattcagg acccacacga cgggaagaca 1741 agttcatgta ctttgagttc cctcagccgt tacctgtgtg tggtgatatc aaagtagagt 1801 tcttccacaa acagaacaag atgctaaaaa aggacaaaat gtttcacttt tgggtaaata 1861 cattcttcat accaggacca gaggaaacct cagaaaaagt agaaaatgga agtctatgtg 1921 atcaagaaat cgatagcatt tgcagtatag agcgtgcaga taatgacaag gaatatctag 1981 tacttacttt aacaaaaaat gatcttgaca aagcaaataa agacaaagcc aaccgatact 2041 tttctccaaa ttttaaggtg aagctgtact tcacaaaaac agtagaggag ccgtcaaatc 2101 cagaggctag cagttcaact tctgtaacac cagatgttag tgacaatgaa cctgatcatt 2161 atagatattc tgacaccact gactctgatc cagagaatga accttttgat gaagatcagc 2221 atacacaaat tacaaaagtc tgaatttttt tttatcaaga gggataaaac accatgaaaa 2281 taaacttgaa taaactgaaa atggaccttt ttttttttaa tggcaatagg acattgtgtc 2341 agattaccag ttataggaac aattctcttt tcctgaccaa tcttgtttta ccctatacat 2401 ccacagggtt ttgacacttg ttgtccagtt gaaaaaaggt tgtgtagctg tgtcatgtat 2461 ataccttttt gtgtcaaaag gacatttaaa attcaattag gattaataaa gatggcactt 2521 tcccgtttta ttccagtttt ataaaaagtg gagacagact gatgtgtata cgtaggaatt 2581 ttttcctttt gtgttctgtc accaactgaa gtggctaaag agctttgtga tatactggtt 2641 cacatcctac ccctttgcac ttgtggcaac agataagttt gcagttggct aagagaggtt 2701 tccgaagggt tttgctacat tctaatgcat gtattcgggt taggggaatg gagggaatgc 2761 tcagaaagga aataatttta tgctggactc tggaccatat accatctcca gctatttaca 2821 cacacctttc tttagcatgc tacagttatt aatctggaca ttcgaggaat tggccgctgt 2881 cactgcttgt tgtttgcgca ttttttttta aagcatattg gtgctagaaa aggcagctaa 2941 aggaagtgaa tctgtattgg ggtacaggaa tgaaccttct gcaacatctt aagatccaca 3001 aatgaaggga tataaaaata atgtcatagg taagaaacac agcaacaatg acttaaccat 3061 ataaatgtgg aggctatcaa caaagaatgg gcttgaaaca ttataaaaat tgacaatgat 3121 ttattaaata tgttttctca attgtaacga cttctccatc tcctgtgtaa tcaaggccag 3181 tgctaaaatt cagatgctgt tagtacctac atcagtcaac aacttacact tattttacta 3241 gttttcaatc ataatacctg ctgtggatgc ttcatgtgct gcctgcaagc ttcttttttc 3301 tcattaaata taaaatattt tgtaatgctg cacagaaatt ttcaatttga gattctacag 3361 taagcgtttt ttttctttga agatttatga tgcacttatt caatagctgt cagccgttcc 3421 acccttttga ccttacacat tctattacaa tgaattttgc agttttgcac attttttaaa 3481 tgtcattaac tgttagggaa ttttacttga atactgaata catataatgt ttatattaaa 3541 aaggacattt gtgttaaaaa ggaaattaga gttgcagtaa actttcaatg ctgcacacaa 3601 aaaaaagaca tttgattttt cagtagaaat tgtcctacat gtgctttatt gatttgctat 3661 tgaaagaata gggttttttt tttttttttt tttttttttt ttaaatgtgc agtgttgaat 3721 catttcttca tagtgctccc ccgagttggg actagggctt caatttcact tcttaaaaaa 3781 aatcatcata tatttgatat gcccagactg catacgattt taagcggagt acaactacta 3841 ttgtaaagct aatgtgaaga tattattaaa aaggtttttt tttccagaaa tttggtgtct 3901 tcaaattata ccttcacctt gacatttgaa tatccagcca ttttgtttct taatggtata 3961 aaattccatt ttcaataact tattggtgct gaaattgttc actagctgtg gtctgaccta 4021 gttaatttac aaatacagat tgaataggac ctactagagc agcatttata gagtttgatg 4081 gcaaatagat taggcagaac ttcatctaaa atattcttag taaataatgt tgacacgttt 4141 tccatacctt gtcagtttca ttcaacaatt tttaaatttt taacaaagct cttaggattt 4201 acacatttat atttaaacat tgatatatag agtattgatt gattgctcat aagttaaatt 4261 ggtaaagtta gagacaacta ttctaacacc tcaccattga aatttatatg ccaccttgtc 4321 tttcataaaa gctgaaaatt gttacctaaa atgaaaatca acttcatgtt ttgaagatag 4381 ttataaatat tgttctttgt tacaatttcg ggcaccgcat attaaaacgt aactttattg 4441 ttccaatatg taacatggag ggccaggtca taaataatga cattataatg ggcttttgca 4501 ctgttattat ttttcctttg gaatgtgaag gtctgaatga gggttttgat tttgaatgtt 4561 tcaatgtttt tgagaagcct tgcttacatt ttatggtgta gtcattggaa atggaaaaat 4621 ggcattatat atattatata tataaatata tattatacat actctcctta ctttatttca 4681 gttaccatcc ccatagaatt tgacaagaat tgctatgact gaaaggtttt cgagtcctaa 4741 ttaaaacttt atttatggca gtattcataa ttagcctgaa atgcattctg taggtaatct 4801 ctgagtttct ggaatatttt cttagacttt ttggatgtgc agcagcttac atgtctgaag 4861 ttacttgaag gcatcacttt taagaaagct tacagttggg ccctgtacca tcccaagtcc 4921 tttgtagctc ctcttgaaca tgtttgccat acttttaaaa gggtagttga ataaatagca 4981 tcaccattct ttgctgtggc acaggttata aacttaagtg gagtttaccg gcagcatcaa 5041 atgtttcagc tttaaaaaat aaaagtaggg tacaagttta atgtttagtt ctagaaattt 5101 tgtgcaatat gttcataacg atggctgtgg ttgccacaaa gtgcctcgtt tacctttaaa 5161 tactgttaat gtgtcatgca tgcagatgga aggggtggaa ctgtgcacta aagtgggggc 5221 tttaactgta gtatttggca gagttgcctt ctacctgcca gttcaaaagt tcaacctgtt 5281 ttcatataga atatatatac taaaaaattt cagtctgtta aacagcctta ctctgattca 5341 gcctcttcag atactcttgt gctgtgcagc agtggctctg tgtgtaaatg ctatgcactg 5401 aggatacaca aaaataccaa tatgatgtgt acaggataat gcctcatccc aatcagatgt 5461 ccatttgtta ttgtgtttgt taacaaccct ttatctctta gtgttataaa ctccacttaa 5521 aactgattaa agtctcattc ttgtcattgt gtgggtgttt tattaaatga gagtttataa 5581 ttcaaattgc ttaagtccat tgaagtttta attaatgggc agccaaatgt gaatacaaag 5641 ttttcagttt ttttttttcc tgctgtcctt caaagcctac tgtttaaaaa aaaaaaaaaa 5701 aaaaaacatg gcctgagagt agagtatctg tctactcatg tttaattaag gaaaaacact 5761 tatttttagg gctttagtca tcacttcata aattgtataa gcacattaaa tagcgttcta 5821 gtcctgaaaa agtccaagat tcttagaaaa ttgtgcatat ttttattatg acagatgttt 5881 gaagataatt ccccagaatg gatttgatac tttagatttc aattttgtgg cttttgtcta 5941 ttattctgta ctctgccatc agcatatgga aagcttcatt tactcatcat gacttgtgcc 6001 atataaaaat tgatatttcg gaatagtcta aaggactttt tgtacttgaa tttaatcatg 6061 ttgtttctaa tattcttaaa agcttgaaga ctaaagcata tcctttcaac aaagcatagt 6121 aaggtaataa gaaagtgtag tttgtacaag tgttaaaaaa ataaagtaga caatgttaca 6181 gtgggactta ttatttcaag tttacatttt ctccatgtaa ttttttaaaa agtaaatgaa 6241 aaaatgtgca ataatgtaaa atatgaagtg tatgtgtaca cacattttat ttttcggtat 6301 cttgggtata cgtatggttg aaaactatac tggagtctaa aagtattcta atttataaga 6361 agacattttg gtgatgtttg aaaaatagaa atgtgctagt tttgttttta tatcatgtcc 6421 tttgtacgtt gtaatatgag ctggcttggt tcagtaaatg ccatcaccat ttccattgag 6481 aatttaaaac tcaccagtgt ttaatatgca ggcttccaaa ggcttatgaa aaaaatcaag 6541 acccttaaat ctagttaatt tgctgctaac atgaaactct ttggttcttt tatttttgcc 6601 agataattag acacacatct aaagcttagt cttaaatggc ttaagtgtag ctattgatta 6661 gtgctgttgc tagttcagaa agaaatgttt gtgaatggaa acaagaatat tcagtccaaa 6721 ctgttgtaag gacagtacct gaaaaccagg aaacaggata atggaaaaag tcttttaaag 6781 atgaaatgtt ggagccaact ttcttataga attaattgta tgtggctata gaaagcctaa 6841 tgattgttgc ttatttttga gagcatatta ttcttttatg accataatct tgctgttttt 6901 ccatcttcca aaagatcttc cttctaatat gtatatcaga atgtgggtag ccagtcagac 6961 aaattcatat tggttggtag ctttaaaaag tttgtaatgt gaagacagga aaggacaaaa 7021 tagtttgctt tggtggtagt actctggttg ttaagctagg tattttgaga ctacttcccc 7081 atcacaacaa caataaaata atcactcata atcctatcac ctggagacat agccatcgtt 7141 aatatgttag tgactataca atcatgtttt cttctgtata tccatgtata ttctttaaaa 7201 atgaaattta tactgtacct gatctcaaag ctttttagct tagtatatct gtcatgaatt 7261 tgtaggatgt tccattgcat cagaaaacgg acagtgattt gattactttc taatgccaca 7321 gatgcagatt acatgtagtt attgagaatc ctttcgaatt cagtggctta atcatgaatg 7381 tctaaatatt gttgacatta ggatgataca tgtaaattaa agttacattt gtttagcata 7441 gacaagctta acattgtaga tgtttctctt caaaaatcat cttaaacatt tgcatttgga 7501 attgtgttaa atagaatgtg tgaaacactg tattagtaaa cttcatcacc tttctacttc 7561 cttatagttt gaacttttca gtttttgtag ttcccaaaca gttgctcaat ttagagcaaa 7621 ttaatttaac acctgccaaa aaaaggctgc tgttggctta tcagttgtct ttaaattcaa 7681 atgctcatgt gacttttatc acatcaaaaa atatttcatt aatgattcac ctttagctct 7741 gaaaattacc gcgtttagta attatagtgg gcttataaaa acatgcaact ctttttgata 7801 gttatttgag aattttggtg aaaaatattt agctgagggc agtatagaac ttataaacca 7861 atatattgat atttttaaaa catttttaca tataagtaaa ctgccatctt tgagcataac 7921 tacatttaaa aataaagctg catattttta aatcaagtgt ttaacaagaa tttatatttt 7981 ttatttttta aaattaaaaa taatttatat ttcctctgtt gcatgaggat tctcatctgt 8041 gcttataatg gttagagatt ttatttgtgt ggaatgaagt gaggcttgta gtcatggttc 8101 tagtgtttca gtttgccaag tctgtttact gcagtgaaat tcatcaaatg tttcagtgtg 8161 gttttctgta gcctatcatt tactggctat ttttttatgt acacctttag gattttctgc 8221 ctactctatc cagttgtcca aatgatatcc tacattttac aaatgccctt tcagtttcta 8281 ttttcttttt ccattaaatt gccctcatgt cctaatgtgc agtttgtaag tgtgtgtgtg 8341 tgtgtctgtg tgtgtgtgaa tttgattttc aagagtgcta gacttccaat ttgagagatt 8401 aaataattta attcaggcaa acatttttca ttggaatttc acagttcatt gtaatgaaaa 8461 tgttaatcct ggatgacctt tgacatacag taatgaatct tggatattaa tgaatttgtt 8521 agtagcatct tgatgtgtgt tttaatgagt tattttcaaa gttgtgcatt aaaccaaagt 8581 tggcatactg gaagtgttta tatcaagttc catttggcta ctgatggaca aaaaatagaa 8641 atgccttcct atggagagta tttttccttt aaaaaattaa aaaggttaat tattttgact 8701 aaaaaaaaaa aaaaaaaa

By "Phosphatase and Tensin Homolog (PTEN) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.

NP_000305, version NP_000305.3, incorporated herein by reference, as reproduced below (SEQ ID NO: 4):

1 mtaiikeivs rnkrryqedg fdldltyiyp niiamgfpae rlegvyrnni dd vrfldsk 61 hknhykiynl caerhydtak fncrvaqypf edhnppqlel ikpfcedldq wlseddnhva 121 aihckagkgr tgvmicayll hrgkflkaqe aldfygevrt rdkkgvtips qrryvyyysy 181 llknhldyrp vallfhkmmf etipmfsggt cnpqf vcql kvkiyssnsg ptrredkfmy 241 fefpqplpvc gdikveffhk qnkmlkkdkm fhfwvntffi pgpeetsekv engslcdqei 301 dsicsierad ndkeylvltl tkndldkank dkanryfspn fkvklyftkt veepsnpeas 361 sstsvtpdvs dnepdhyrys dttdsdpene pfdedqhtqi tkv

By "Tuberous Sclerosis 1 (TSC1) nucleic acid molecule" is meant a polynucleotide encoding a TSC1 polypeptide. An exemplary TSC1 nucleic acid molecule is provided at NCBI Accession No. NM_000368, version NM_000368.4, incorporated herein by reference, and reproduced below (SEQ ID NO: 5):

1 acgacggggg aggtgctgta cgtccaagat ggcggcgccc tgtaggctgg agggactgtg

61 aggtaaacag ctgaggggga ggagacggtg gtgaccatga aagacaccag gttgacagca 121 ctggaaactg aagtaccagt tgtcgctaga acagtttggt agtggcccca atgaagaacc 181 ttcagaacct gtagcacacg tcctggagcc agcacagcgc cttcgagcga gagaatggcc 241 caacaagcaa atgtcgggga gcttcttgcc atgctggact cccccatgct gggtgtgcgg 301 gacgacgtga cagctgtctt taaagagaac ctcaattctg accgtggccc tatgcttgta 361 aacaccttgg tggattatta cctggaaacc agctctcagc cggcattgca catcctgacc 421 accttgcaag agccacatga caagcacctc ttggacagga ttaacgaata tgtgggcaaa 481 gccgccactc gtttatccat cctctcgtta ctgggtcatg tcataagact gcagccatct 541 tggaagcata agctctctca agcacctctt ttgccttctt tactaaaatg tctcaagatg 601 gacactgacg tcgttgtcct cacaacaggc gtcttggtgt tgataaccat gctaccaatg 661 attccacagt ctgggaaaca gcatcttctt gatttctttg acatttttgg ccgtctgtca 721 tcatggtgcc tgaagaaacc aggccacgtg gcggaagtct atctcgtcca tctccatgcc 781 agtgtgtacg cactctttca tcgcctttat ggaatgtacc cttgcaactt cgtctccttt 841 ttgcgttctc attacagtat gaaagaaaac ctggagactt ttgaagaagt ggtcaagcca 901 atgatggagc atgtgcgaat tcatccggaa ttagtgactg gatccaagga ccatgaactg 961 gaccctcgaa ggtggaagag attagaaact catgatgttg tgatcgagtg tgccaaaatc 1021 tctctggatc ccacagaagc ctcatatgaa gatggctatt ctgtgtctca ccaaatctca 1081 gcccgctttc ctcatcgttc agccgatgtc accaccagcc cttatgctga cacacagaat 1141 agctatgggt gtgctacttc taccccttac tccacgtctc ggctgatgtt gttaaatatg 1201 ccagggcagc tacctcagac tctgagttcc ccatcgacac ggctgataac tgaaccacca 1261 caagctactc tttggagccc atctatggtt tgtggtatga ccactcctcc aacttctcct 1321 ggaaatgtcc cacctgatct gtcacaccct tacagtaaag tctttggtac aactgcaggt 1381 ggaaaaggaa ctcctctggg aaccccagca acctctcctc ctccagcccc actctgtcat 1441 tcggatgact acgtgcacat ttcactcccc caggccacag tcacaccccc caggaaggaa 1501 gagagaatgg attctgcaag accatgtcta cacagacaac accatcttct gaatgacaga 1561 ggatcagaag agccacctgg cagcaaaggt tctgtcactc taagtgatct tccagggttt 1621 ttaggtgatc tggcctctga agaagatagt attgaaaaag ataaagaaga agctgcaata 1681 tctagagaac tttctgagat caccacagca gaggcagagc ctgtggttcc tcgaggaggc 1741 tttgactctc ccttttaccg agacagtctc ccaggttctc agcggaagac ccactcggca 1801 gcctccagtt ctcagggcgc cagcgtgaac cctgagcctt tacactcctc cctggacaag 1861 cttgggcctg acacaccaaa gcaagccttt actcccatag acctgccctg cggcagtgct 1921 gatgaaagcc ctgcgggaga cagggaatgc cagacttctt tggagaccag tatcttcact 1981 cccagtcctt gtaaaattcc acctccgacg agagtgggct ttggaagcgg gcagcctccc 2041 ccgtatgatc atctttttga ggtggcattg ccaaagacag cccatcattt tgtcatcagg 2101 aagactgagg agctgttaaa gaaagcaaaa ggaaacacag aggaagatgg tgtgccctct 2161 acctccccaa tggaagtgct ggacagactg atacagcagg gagcagacgc gcacagcaag 2221 gagctgaaca agttgccttt acccagcaag tctgtcgact ggacccactt tggaggctct 2281 cctccttcag atgagatccg caccctccga gaccagttgc ttttactgca caaccagtta 2341 ctctatgagc gttttaagag gcagcagcat gccctccgga acaggcggct cctccgcaag 2401 gtgatcaaag cagcagctct ggaggaacat aatgctgcca tgaaagatca gttgaagtta 2461 caagagaagg acatccagat gtggaaggtt agtctgcaga aagaacaagc tagatacaat 2521 cagctccagg agcagcgtga cactatggta accaagctcc acagccagat cagacagctg 2581 cagcatgacc gagaggaatt ctacaaccag agccaggaat tacagacgaa gctggaggac 2641 tgcaggaaca tgattgcgga gctgcggata gaactgaaga aggccaacaa caaggtgtgt 2701 cacactgagc tgctgctcag tcaggtttcc caaaagctct caaacagtga gtcggtccag 2761 cagcagatgg agttcttgaa caggcagctg ttggttcttg gggaggtcaa cgagctctat 2821 ttggaacaac tgcagaacaa gcactcagat accacaaagg aagtagaaat gatgaaagcc 2881 gcctatcgga aagagctaga aaaaaacaga agccatgttc tccagcagac tcagaggctt 2941 gatacctccc aaaaacggat tttggaactg gaatctcacc tggccaagaa agaccacctt 3001 cttttggaac agaagaaata tctagaggat gtcaaactcc aggcaagagg acagctgcag 3061 gccgcagaga gcaggtatga ggctcagaaa aggataaccc aggtgtttga attggagatc 3121 ttagatttat atggcaggtt ggagaaagat ggcctcctga aaaaacttga agaagaaaaa 3181 gcagaagcag ctgaagcagc agaagaaagg cttgactgtt gtaatgacgg gtgctcagat 3241 tccatggtag ggcacaatga agaggcatct ggccacaacg gtgagaccaa gacccccagg 3301 cccagcagcg cccggggcag tagtggaagc agaggtggtg gaggcagcag cagcagcagc 3361 agcgagcttt ctaccccaga gaaaccccca caccagaggg caggcccatt cagcagtcgg 3421 tgggagacga ctatgggaga agcgtctgcc agcatcccca ccactgtggg ctcacttccc 3481 agttcaaaaa gcttcctggg tatgaaggct cgagagttat ttcgtaataa gagcgagagc 3541 cagtgtgatg aggacggcat gaccagtagc ctttctgaga gcctaaagac agaactgggc 3601 aaagacttgg gtgtggaagc caagattccc ctgaacctag atggccctca cccgtctccc 3661 ccgaccccgg acagtgttgg acagctacat atcatggact acaatgagac tcatcatgaa 3721 cacagctaag gaatgatggt caatcagtgt taacttgcat attgttggca cagaacagga 3781 ggtgtgaatg cacgtttcaa agctttcctg tttccagggt ctgagtgcaa gttcatgtgt 3841 ggaaatggga cggaggtcct ttggacagct gactgaatgc agaacggttt ttggatctgg 3901 cattgaaatg cctcttgacc ttcccctcca cccgccctaa ccccctctca tttacctcgc 3961 agtgtgttct aatccaaggg ccagttggtg ttcctcagta gctttacttt cttcctttcc 4021 cccccaaatg gttgcgtcct ttgaacctgt gcaatatgag gccaaattta atctttgagt 4081 ctaacacacc actttctgct ttcccgaagt tcagataact gggttggctc tcaattagac 4141 caggtagttt gttgcattgc aggtaagtct ggttttgtcc cttccaggag gacatagcct 4201 gcaaagctgg ttgtctttac atgaaagcgt ttacatgaga ctttccgact gcttttttga 4261 ttctgaagtt cagcatctaa agcagcaggt ctagaagaac aacggtttat tcatacttgc 4321 attcttttgg cagttctgat aagcttccta gaaagttctg tgtaaacaga agcctgtttc 4381 agaaatctgg agctggcact gtggagacca cacacccttt gggaaagctc ttgtctcttc 4441 ttcccccact acctcttatt tatttggtgt ttgcttgaat gctggtacta ttgtgaccac 4501 aggctggtgt gtaggtggta aaacctgttc tccataggag ggaaggagca gtcactggga 4561 gaggttaccc gagaagcact tgagcatgag gaactgcacc tttaggccat ctcagcttgc 4621 tgggcctttt gttaaaccct tctgtctact ggcctccctt tgtgtgcata cgcctcttgt 4681 tcatgtcagc ttatatgtga cactgcagca gaaaggctct gaaggtccaa agagtttctg 4741 caaagtgtat gtgaccatca tttcccaggc cattagggtt gcctcactgt agcaggttct 4801 aggctaccag aagaggggca gctttttcat accaattcca actttcaggg gctgactctc 4861 cagggagctg atgtcatcac actctccatg ttagtaatgg cagagcagtc taaacagagt 4921 ccgggagaat gctggcaaag gctggctgtg tatacccact aggctgcccc acgtgctccc 4981 gagagatgac actagtcaga aaattggcag tggcagagaa tccaaactca acaagtgctc 5041 ctgaaagaaa cgctagaagc ctaagaactg tggtctggtg ttccagctga ggcaggggga 5101 tttggtagga aggagccagt gaacttggct ttcctgtttc tatctttcat taaaaagaat 5161 agaaggattc agtcataaag aggtaaaaaa ctgtcacggt acgaaatctt agtgcccacg 5221 gaggcctcga gcagagagaa tgaaagtctt tttttttttt tttttttttt agcatggcaa 5281 taaatattct agcatcccta actaaagggg actagacagt tagagactct gtcaccctag 5341 ctataccagc agaaaacctg ttcaggcagg ctttctgggt gtgactgatt cccagcctgt 5401 ggcagggcgt ggtcccaact actcagccta gcacaggctg gcagttggta ctgaattgtc 5461 agatgtggag tattagtgac accacacatt taattcagct ttgtccaaag gaaagcttaa 5521 aacccaatac agtctagttt cctggttccg ttttagaaaa ggaaaacgtg aacaaactta 5581 gaaagggaag gaaatcccat cagtgaatcc tgaaactggt tttaagtgct ttccttctcc 5641 tcatgcccaa gagatctgtg ccatagaaca agataccagg cacttaaagc cttttcctga 5701 attggaaagg aaaagaggcc caagtgcaaa agaaaaaaca ttttagaaac ggacagctta 5761 taaaaataaa gggaagaaag gaggcagcat ggagagaggc ctgtgctaga agctccatgg 5821 acgtgtctgc acagggtcct cagctcatcc atgcggcctg ggtgtccttt tactcagctt 5881 tataacaaat gtggctccaa gctcaggtgc ctttgagttc taggaggctg tgggttttat 5941 tcaactacgg ttgggagaat gagacctgga gtcatgttga aggtgcccaa cctaaaaatg 6001 taggctttca tgttgcaaag aactccagag tcagtagtta ggtttggttt ggttttggac 6061 atgataaacc tgccaagagt caacaggtca cttgatcatg ctgcagtggg tagttctaag 6121 gatggaaagg tgacagtatt actctcgaga ggcaattcag tcctgggcaa aggtattagt 6181 acaataagcg ttaagggcag agtctacctt gaaaccaatt aagcagcttg gtattcataa 6241 atattgggat tggatggcct ccatccagaa atcactatgg gtgagcatac ctgtctcagc 6301 tgtttggcca atgtgcataa cctactcgga tccccacctg acactaacca gagtcagcac 6361 aggccccgag gagcccgaag tctgctgctg tgcagcatgg aattccttta aaaaggtgca 6421 ctacagtttt agcggggagg gggataggaa gacgcagagc aaatgagctc cggagtccct 6481 gcaggtgaat aaacacacag atctgcatct gatagaactt tgatggattt tcaaaaagcc 6541 gttgacaagg ctctgctata cagtctataa aaattgttat tatgggattg gaagaaacac 6601 gtggtcatga atagaaaaaa aacaaaccca aaggtaggaa ggtcaaggtc atttcttaga 6661 tggagaagtt gtgaaagatg tccttggaga tgagttttag gaccagcatt actaaggcag 6721 gtgggcagac agtgacctct ctaggtgtgt ccacagagtt tttcaggaga gaaaactgcc 6781 tgacctttgg gactaagctg cggaatcttc ttactaagct tgaagagtgg agaggcgaga 6841 ggtgagctac tttgtgagcc aaagcttatg tgacatggtt ggggaaacag tccaaactgt 6901 tctgagaagg tgaactgtta cgacccagga caattagaaa aattcaccca ccatgccgca 6961 cattactggg taaaagcagg gcagcaggga acaaaactcc agactcttgg gccgtcccca 7021 tttgcaacag cacacatagt ttctggtata tttgttggga aagataaaac tctagcagtt 7081 gttgagggga ggatgtataa aatggtcatg gggatgaaag gatctctgag accacagagg 7141 ctcagactca ctgttaagaa tagaaaactg ggtatgcgtt tcatgtagcc agcagaactg 7201 aagtgtgctg tgacaagcca atgtgaattt ctaccaaata gtagagcata ccacttgaag 7261 aaggaaagaa ccgaagagca aacaaaagtt ctgcgtaatg agactcacct tttctcgctg 7321 aaagcactaa gaggtgggag gaggcctgca caggctggag gagggtttgg gcagagcgaa 7381 gacccggcca ggaccttggt gagatggggt gccgcccacc tcctgcggat actcttggag 7441 agttgttccc ccagggggct ctgccccacc tggagaagga agctgcctgg tgtggagtga 7501 ctcaaatcag tatacctatc tgctgcacct tcactctcca gggtacatgc tttaaaaccg 7561 acccgcaaca agtattggaa aaatgtatcc agtctgaaga tgtttgtgta tctgtttaca 7621 tccagagttc tgtgacacat gccccccaga ttgctgcaaa gatcccaagg cattgattgc 7681 acttgattaa gcttttgtct gtaggtgaaa gaacaagttt aggtcgagga ctggccccta 7741 ggctgctgct gtgacccttg tcccatgtgg cttgtttgcc tgtccgggac tcttcgatgt 7801 gcccagggga gcgtgttcct gtctcttcca tgccgtcctg cagtccttat ctgctcgcct 7861 gagggaagag tagctgtagc tacaagggaa gcctgcctgg aagagccgag cacctgtgcc 7921 catggcttct ggtcatgaaa cgagttaatg atggcagagg agcttcctcc ccacttcgca 7981 gcgccacatt atccatcctc tgagataagt aggctggttt aaccattgga atggaccttt 8041 cagtggaaac cctgagagtc tgagaacccc cagaccaacc cttccctccc tttccccacc 8101 tcttacagtg tttggacagg agggtatggt gctgctctgt gtagcaagta ctttggctta 8161 tgaaagaggc agccacgcat tttgcactag gaagaatcag taatcacttt tcagaagact 8221 tctatggacc acaaatatat tacggaggaa cagattttgc taagacataa tctagtttta 8281 taactcaatc atgaatgaac catgtgtggc aaacttgcag tttaaagggg tcccatcagt 8341 gaaagaaact gatttttttt aacggactgc ttttagttaa attgaagaaa gtcagctctt 8401 gtcaaaaggt ctaaactttc ccgcctcaat cctaaaagca tgtcaacaat ccacatcaga 8461 tgccataaat atgaactgca ggataaaatg gtacaatctt agtgaatggg aattggaatc 8521 aaaagagttt gctgtccttc ttagaatgtt ctaaaatgtc aaggcagttg cttgtgttta 8581 actgtgaaca aataaaaatt tattgttttg cactacaaaa aaaaaa

By "Tuberous Sclerosis 1 (TSC1) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_000359, version NP 000359.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 6):

1 maqqanvgel lamldspmlg vrddvtavfk enlnsdrgpm lvntlvdyyl etssqpalhi

61 lttlqephdk hlldrineyv gkaatrlsil sllghvirlq pswkhklsqa pllpsllkcl 121 kmdtdv vlt tgvlvlitml pmipqsgkqh lldffdifgr lsswclkkpg hvaevylvhl 181 hasvyalfhr lygmypcnf s flrshysmk enletfeevv kpmmehvrih pelvtgskdh 241 eldprrwkrl ethd vieca kisldpteas yedgysvshq isarfphrsa dvttspyadt 301 qnsygcatst pystsrlmll nmpgqlpqtl sspstrlite ppqatlwsps mvcgmttppt 361 spgnvppdls hpyskvfgtt aggkgtplgt patspppapl chsddyvhis lpqatvtppr 421 keermdsarp clhrqhhlln drgseeppgs kgs tlsdlp gflgdlasee dsiekdkeea 481 aisrelseit taeaep vpr ggfdspfyrd slpgsqrkth saasssqgas vnpeplhssl 541 dklgpdtpkq aftpidlpcg sadespagdr ecqtsletsi ftpspckipp ptrvgfgsgq 601 pppydhlfev alpktahhf irkteellkk akgnteedgv pstspmevld rliqqgadah 661 skelnklplp sksvdwthfg gsppsdeirt lrdqllllhn qllyerfkrq qhalrnrrll 721 rkvikaaale ehnaamkdql klqekdiqmw kvslqkeqar ynqlqeqrdt mvtklhsqir 781 qlqhdreefy nqsqelqtkl edcrnmiael rielkkannk vchtelllsq vsqklsnses 841 vqqqmeflnr qllvlgevne lyleqlqnkh sdttkevemm kaayrkelek nrshvlqqtq 901 rldtsqkril eleshlakkd hllleqkkyl edvklqargq lqaaesryea qkritqvfel 961 eildlygrle kdgllkklee ekaeaaeaae erldccndgc sdsmvghnee asghngetkt 1021 prpssargss gsrggggsss ssselstpek pphqragpfs srwettmgea sasipttvgs 1081 lpssksflgm karelfrnks esqcdedgmt sslseslkte lgkdlgveak iplnldgphp 1141 spptpdsvgq lhimdyneth hehs

By "Neurofibromin 1 (NF1) nucleic acid molecule" is meant a polynucleotide encoding a NF1 polypeptide. An exemplary NF1 nucleic acid molecule is provided at NCBI Accession No. NM_001042492, version NM_001042492.2, incorporated herein by reference, and reproduced below (SEQ ID NO: 7):

1 aatctctagc tcgctcgcgc tccctctccc cgggccgtgg aaaggatccc acttccggtg 61 gggtgtcatg gcggcgtctc ggactgtgat ggctgtgggg agacggcgct agtggggaga 121 gcgaccaaga ggccccctcc cctccccggg tccccttccc ctatccccct ccccccagcc 181 tccttgccaa cgcccccttt ccctctcccc ctcccgctcg gcgctgaccc cccatcccca 241 cccccgtggg aacactggga gcctgcactc cacagaccct ctccttgcct cttccctcac 301 ctcagcctcc gctccccgcc ctcttcccgg cccagggcgc cggcccaccc ttccctccgc 361 cgccccccgg ccgcggggag gacatggccg cgcacaggcc ggtggaatgg gtccaggccg 421 tggtcagccg cttcgacgag cagcttccaa taaaaacagg acagcagaac acacatacca 481 aagtcagtac tgagcacaac aaggaatgtc taatcaatat ttccaaatac aagttttctt 541 tggttataag cggcctcact actattttaa agaatgttaa caatatgaga atatttggag 601 aagctgctga aaaaaattta tatctctctc agttgattat attggataca ctggaaaaat 661 gtcttgctgg gcaaccaaag gacacaatga gattagatga aacgatgctg gtcaaacagt 721 tgctgccaga aatctgccat tttcttcaca cctgtcgtga aggaaaccag catgcagctg 781 aacttcggaa ttctgcctct ggggttttat tttctctcag ctgcaacaac ttcaatgcag 841 tctttagtcg catttctacc aggttacagg aattaactgt ttgttcagaa gacaatgttg 901 atgttcatga tatagaattg ttacagtata tcaatgtgga ttgtgcaaaa ttaaaacgac 961 tcctgaagga aacagcattt aaatttaaag ccctaaagaa ggttgcgcag ttagcagtta 1021 taaatagcct ggaaaaggca ttttggaact gggtagaaaa ttatccagat gaatttacaa 1081 aactgtacca gatcccacag actgatatgg ctgaatgtgc agaaaagcta tttgacttgg 1141 tggatggttt tgctgaaagc accaaacgta aagcagcagt ttggccacta caaatcattc 1201 tccttatctt gtgtccagaa ataatccagg atatatccaa agacgtggtt gatgaaaaca 1261 acatgaataa gaagttattt ctggacagtc tacgaaaagc tcttgctggc catggaggaa 1321 gtaggcagct gacagaaagt gctgcaattg cctgtgtcaa actgtgtaaa gcaagtactt 1381 acatcaattg ggaagataac tctgtcattt tcctacttgt tcagtccatg gtggttgatc 1441 ttaagaacct gctttttaat ccaagtaagc cattctcaag aggcagtcag cctgcagatg 1501 tggatctaat gattgactgc cttgtttctt gctttcgtat aagccctcac aacaaccaac 1561 actttaagat ctgcctggct cagaattcac cttctacatt tcactatgtg ctggtaaatt 1621 cactccatcg aatcatcacc aattccgcat tggattggtg gcctaagatt gatgctgtgt 1681 attgtcactc ggttgaactt cgaaatatgt ttggtgaaac acttcataaa gcagtgcaag 1741 gttgtggagc acacccagca atacgaatgg caccgagtct tacatttaaa gaaaaagtaa 1801 caagccttaa atttaaagaa aaacctacag acctggagac aagaagctat aagtatcttc 1861 tcttgtccat ggtgaaacta attcatgcag atccaaagct cttgctttgt aatccaagaa 1921 aacaggggcc cgaaacccaa ggcagtacag cagaattaat tacagggctc gtccaactgg 1981 tccctcagtc acacatgcca gagattgctc aggaagcaat ggaggctctg ctggttcttc 2041 atcagttaga tagcattgat ttgtggaatc ctgatgctcc tgtagaaaca ttttgggaga 2101 ttagctcaca aatgcttttt tacatctgca agaaattaac tagtcatcaa atgcttagta 2161 gcacagaaat tctcaagtgg ttgcgggaaa tattgatctg caggaataaa tttcttctta 2221 aaaataagca ggcagataga agttcctgtc actttctcct tttttacggg gtaggatgtg 2281 atattccttc tagtggaaat accagtcaaa tgtccatgga tcatgaagaa ttactacgta 2341 ctcctggagc ctctctccgg aagggaaaag ggaactcctc tatggatagt gcagcaggat 2401 gcagcggaac ccccccgatt tgccgacaag cccagaccaa actagaagtg gccctgtaca 2461 tgtttctgtg gaaccctgac actgaagctg ttctggttgc catgtcctgt ttccgccacc 2521 tctgtgagga agcagatatc cggtgtgggg tggatgaagt gtcagtgcat aacctcttgc 2581 ccaactataa cacattcatg gagtttgcct ctgtcagcaa tatgatgtca acaggaagag 2641 cagcacttca gaaaagagtg atggcactgc tgaggcgcat tgagcatccc actgcaggaa 2701 acactgaggc ttgggaagat acacatgcaa aatgggaaca agcaacaaag ctaatcctta 2761 actatccaaa agccaaaatg gaagatggcc aggctgctga aagccttcac aagaccattg 2821 ttaagaggcg aatgtcccat gtgagtggag gaggatccat agatttgtct gacacagact 2881 ccctacagga atggatcaac atgactggct tcctttgtgc ccttggggga gtgtgcctcc 2941 agcagagaag caattctggc ctggcaacct atagcccacc catgggtcca gtcagtgaac 3001 gtaagggttc tatgatttca gtgatgtctt cagagggaaa cgcagataca cctgtcagca 3061 aatttatgga tcggctgttg tccttaatgg tgtgtaacca tgagaaagtg ggacttcaaa 3121 tacggaccaa tgttaaggat ctggtgggtc tagaattgag tcctgctctg tatccaatgc 3181 tatttaacaa attgaagaat accatcagca agttttttga ctcccaagga caggttttat 3241 tgactgatac caatactcaa tttgtagaac aaaccatagc tataatgaag aacttgctag 3301 ataatcatac tgaaggcagc tctgaacatc tagggcaagc tagcattgaa acaatgatgt 3361 taaatctggt caggtatgtt cgtgtgcttg ggaatatggt ccatgcaatt caaataaaaa 3421 cgaaactgtg tcaattagtt gaagtaatga tggcaaggag agatgacctc tcattttgcc 3481 aagagatgaa atttaggaat aagatggtag aatacctgac agactgggtt atgggaacat 3541 caaaccaagc agcagatgat gatgtaaaat gtcttacaag agatttggac caggcaagca 3601 tggaagcagt agtttcactt ctagctggtc tccctctgca gcctgaagaa ggagatggtg 3661 tggaattgat ggaagccaaa tcacagttat ttcttaaata cttcacatta tttatgaacc 3721 ttttgaatga ctgcagtgaa gttgaagatg aaagtgcgca aacaggtggc aggaaacgtg 3781 gcatgtctcg gaggctggca tcactgaggc actgtacggt ccttgcaatg tcaaacttac 3841 tcaatgccaa cgtagacagt ggtctcatgc actccatagg cttaggttac cacaaggatc 3901 tccagacaag agctacattt atggaagttc tgacaaaaat ccttcaacaa ggcacagaat 3961 ttgacacact tgcagaaaca gtattggctg atcggtttga gagattggtg gaactggtca 4021 caatgatggg tgatcaagga gaactcccta tagcgatggc tctggccaat gtggttcctt 4081 gttctcagtg ggatgaacta gctcgagttc tggttactct gtttgattct cggcatttac 4141 tctaccaact gctctggaac atgttttcta aagaagtaga attggcagac tccatgcaga 4201 ctctcttccg aggcaacagc ttggccagta aaataatgac attctgtttc aaggtatatg 4261 gtgctaccta tctacaaaaa ctcctggatc ctttattacg aattgtgatc acatcctctg 4321 attggcaaca tgttagcttt gaagtggatc ctaccaggtt agaaccatca gagagccttg 4381 aggaaaacca gcggaacctc cttcagatga ctgaaaagtt cttccatgcc atcatcagtt 4441 cctcctcaga attcccccct caacttcgaa gtgtgtgcca ctgtttatac caggcaactt 4501 gccactccct actgaataaa gctacagtaa aagaaaaaaa ggaaaacaaa aaatcagtgg 4561 ttagccagcg tttccctcag aacagcatcg gtgcagtagg aagtgccatg ttcctcagat 4621 ttatcaatcc tgccattgtc tcaccgtatg aagcagggat tttagataaa aagccaccac 4681 ctagaatcga aaggggcttg aagttaatgt caaagatact tcagagtatt gccaatcatg 4741 ttctcttcac aaaagaagaa catatgcggc ctttcaatga ttttgtgaaa agcaactttg 4801 atgcagcacg caggtttttc cttgatatag catctgattg tcctacaagt gatgcagtaa 4861 atcatagtct ttccttcata agtgacggca atgtgcttgc tttacatcgt ctactctgga 4921 acaatcagga gaaaattggg cagtatcttt ccagcaacag ggatcataaa gctgttggaa 4981 gacgaccttt tgataagatg gcaacacttc ttgcatacct gggtcctcca gagcacaaac 5041 ctgtggcaga tacacactgg tccagcctta accttaccag ttcaaagttt gaggaattta 5101 tgactaggca tcaggtacat gaaaaagaag aattcaaggc tttgaaaacg ttaagtattt 5161 tctaccaagc tgggacttcc aaagctggga atcctatttt ttattatgtt gcacggaggt 5221 tcaaaactgg tcaaatcaat ggtgatttgc tgatatacca tgtcttactg actttaaagc 5281 catattatgc aaagccatat gaaattgtag tggaccttac ccataccggg cctagcaatc 5341 gctttaaaac agactttctc tctaagtggt ttgttgtttt tcctggcttt gcttacgaca 5401 acgtctccgc agtctatatc tataactgta actcctgggt cagggagtac accaagtatc 5461 atgagcggct gctgactggc ctcaaaggta gcaaaaggct tgttttcata gactgtcctg 5521 ggaaactggc tgagcacata gagcatgaac aacagaaact acctgctgcc accttggctt 5581 tagaagagga cctgaaggta ttccacaatg ctctcaagct agctcacaaa gacaccaaag 5641 tttctattaa agttggttct actgctgtcc aagtaacttc agcagagcga acaaaagtcc 5701 tagggcaatc agtctttcta aatgacattt attatgcttc ggaaattgaa gaaatctgcc 5761 tagtagatga gaaccagttc accttaacca ttgcaaacca gggcacgccg ctcaccttca 5821 tgcaccagga gtgtgaagcc attgtccagt ctatcattca tatccggacc cgctgggaac 5881 tgtcacagcc cgactctatc ccccaacaca ccaagattcg gccaaaagat gtccctggga 5941 cactgctcaa tatcgcatta cttaatttag gcagttctga cccgagttta cggtcagctg 6001 cctataatct tctgtgtgcc ttaacttgta cctttaattt aaaaatcgag ggccagttac 6061 tagagacatc aggtttatgt atccctgcca acaacaccct ctttattgtc tctattagta 6121 agacactggc agccaatgag ccacacctca cgttagaatt tttggaagag tgtatttctg 6181 gatttagcaa atctagtatt gaattgaaac acctttgttt ggaatacatg actccatggc 6241 tgtcaaatct agttcgtttt tgcaagcata atgatgatgc caaacgacaa agagttactg 6301 ctattcttga caagctgata acaatgacca tcaatgaaaa acagatgtac ccatctattc 6361 aagcaaaaat atggggaagc cttgggcaga ttacagatct gcttgatgtt gtactagaca 6421 gtttcatcaa aaccagtgca acaggtggct tgggatcaat aaaagctgag gtgatggcag 6481 atactgctgt agctttggct tctggaaatg tgaaattggt ttcaagcaag gttattggaa 6541 ggatgtgcaa aataattgac aagacatgct tatctccaac tcctacttta gaacaacatc 6601 ttatgtggga tgatattgct attttagcac gctacatgct gatgctgtcc ttcaacaatt 6661 cccttgatgt ggcagctcat cttccctacc tcttccacgt tgttactttc ttagtagcca 6721 caggtccgct ctcccttaga gcttccacac atggactggt cattaatatc attcactctc 6781 tgtgtacttg ttcacagctt cattttagtg aagagaccaa gcaagttttg agactcagtc 6841 tgacagagtt ctcattaccc aaattttact tgctgtttgg cattagcaaa gtcaagtcag 6901 ctgctgtcat tgccttccgt tccagttacc gggacaggtc attctctcct ggctcctatg 6961 agagagagac ttttgctttg acatccttgg aaacagtcac agaagctttg ttggagatca 7021 tggaggcatg catgagagat attccaacgt gcaagtggct ggaccagtgg acagaactag 7081 ctcaaagatt tgcattccaa tataatccat ccctgcaacc aagagctctt gttgtctttg 7141 ggtgtattag caaacgagtg tctcatgggc agataaagca gataatccgt attcttagca 7201 aggcacttga gagttgctta aaaggacctg acacttacaa cagtcaagtt ctgatagaag 7261 ctacagtaat agcactaacc aaattacagc cacttcttaa taaggactcg cctctgcaca 7321 aagccctctt ttgggtagct gtggctgtgc tgcagcttga tgaggtcaac ttgtattcag 7381 caggtaccgc acttcttgaa caaaacctgc atactttaga tagtctccgt atattcaatg 7441 acaagagtcc agaggaagta tttatggcaa tccggaatcc tctggagtgg cactgcaagc 7501 aaatggatca ttttgttgga ctcaatttca actctaactt taactttgca ttggttggac 7561 accttttaaa agggtacagg catccttcac ctgctattgt tgcaagaaca gtcagaattt 7621 tacatacact actaactctg gttaacaaac acagaaattg tgacaaattt gaagtgaata 7681 cacagagcgt ggcctactta gcagctttac ttacagtgtc tgaagaagtt cgaagtcgct 7741 gcagcctaaa acatagaaag tcacttcttc ttactgatat ttcaatggaa aatgttccta 7801 tggatacata tcccattcat catggtgacc cttcctatag gacactaaag gagactcagc 7861 catggtcctc tcccaaaggt tctgaaggat accttgcagc cacctatcca actgtcggcc 7921 agaccagtcc ccgagccagg aaatccatga gcctggacat ggggcaacct tctcaggcca 7981 acactaagaa gttgcttgga acaaggaaaa gttttgatca cttgatatca gacacaaagg 8041 ctcctaaaag gcaagaaatg gaatcaggga tcacaacacc ccccaaaatg aggagagtag 8101 cagaaactga ttatgaaatg gaaactcaga ggatttcctc atcacaacag cacccacatt 8161 tacgtaaagt ttcagtgtct gaatcaaatg ttctcttgga tgaagaagta cttactgatc 8221 cgaagatcca ggcgctgctt cttactgttc tagctacact ggtaaaatat accacagatg 8281 agtttgatca acgaattctt tatgaatact tagcagaggc cagtgttgtg tttcccaaag 8341 tctttcctgt tgtgcataat ttgttggact ctaagatcaa caccctgtta tcattgtgcc 8401 aagatccaaa tttgttaaat ccaatccatg gaattgtgca gagtgtggtg taccatgaag 8461 aatccccacc acaataccaa acatcttacc tgcaaagttt tggttttaat ggcttgtggc 8521 ggtttgcagg accgttttca aagcaaacac aaattccaga ctatgctgag cttattgtta 8581 agtttcttga tgccttgatt gacacgtacc tgcctggaat tgatgaagaa accagtgaag 8641 aatccctcct gactcccaca tctccttacc ctcctgcact gcagagccag cttagtatca 8701 ctgccaacct taacctttct aattccatga cctcacttgc aacttcccag cattccccag 8761 gaatcgacaa ggagaacgtt gaactctccc ctaccactgg ccactgtaac agtggacgaa 8821 ctcgccacgg atccgcaagc caagtgcaga agcaaagaag cgctggcagt ttcaaacgta 8881 atagcattaa gaagatcgtg tgaagcttgc ttgctttctt ttttaaaatc aacttaacat 8941 gggctcttca ctagtgaccc cttccctgtc cttgcccttt ccccccatgt tgtaatgctg 9001 cacttcctgt tttataatga acccatccgg tttgccatgt tgccagatga tcaactcttc 9061 gaagccttgc ctaaatttaa tgctgccttt tctttaactt tttttcttct acttttggcg 9121 tgtatctggt atatgtaagt gttcagaaca actgcaaaga aagtgggagg tcaggaaact 9181 tttaactgag aaatctcaat tgtaagagag gatgaattct tgaatactgc tactactggc 9241 cagtgatgaa agccatttgc acagagctct gccttctgtg gttttccctt cttcatccta 9301 cagagtaaag tgttagtcct atttatacat ttttcaagat acaagtttat gagagaaata 9361 gtattataac cccagtatgt ttaatctttt agctgtggac ttttttttta accgtacaaa 9421 actgaaagaa ccatagaggt caagcctcag tgacttgaca ccataaagcc acagacaagg 9481 tacttggggg ggagggcagg gaaatttcat attttatagt ggattcttaa gaaatactaa 9541 cacttgagta ttagcaataa ttacaggaaa ataagtgcga ccacatatat cttaacatta 9601 ctgaattaaa actatggctt ctaagtcctt atccaaactc agtcatccaa actagtttat 9661 ttttttctcc agttgattat cttttaattt ttaattttgc taaaggtggt ttttttgtgt 9721 tttgtttttt gtaaaccaaa actatactaa gtatagtaat tatatatata tatatatttt 9781 ttcccctccc cctcttcttt cctaactaat tctgagcagg gtaatcagtg aacaaagtgt 9841 tgaaaattgt tcccagaagg taattttcat agatgtttgc attagctcca tagcaaaatg 9901 gaatggtacg tgacatttag ggtagctgat atttttattt tgttaaataa tttccaagaa 9961 tagagtatgg tgtatattat aaatttcttt gataagatgt attttgaatg tcttttaatc 10021 ttcctcctcc tctccaaaaa aatcagaaac ctctttaaga aaacatgtag gttatatatg 10081 ctagaattgc atttaatcac tgtgaaaaga ctggtcagcc tgcattagta tgacagtagg 10141 ggggctgtta gaattgctgc tatactggtg gtatggatta tcatggcatt ggaattttca 10201 tagtaatgca gatccaattt ctttgtggta cctgcagttt acaaaataat ttgacttcag 10261 tgagcatatt ggtatctgga tgttccaatt tagaactaaa ccatatttat tacaaaaaga 10321 tattaatccc tctactccca ggttcccttt atatgttaag atataatggc tttgaggggg 10381 gaaaaaataa acctagggga gaggggagtt tcctgtagtg ctgtttcatt agaggatttc 10441 agtaaattaa attccacagc taattcaata aataatggta catttaagtg ttctgatttt 10501 aataatatat ttcacattta tccacacagt aacaatgtaa tatgttaatg taaataaaat 10561 tggttttgat actcagaaat aacaagaatt taatttttta aatttgttta cagtcctggg 10621 aaaagtaaga attatttgcc aaaataagag gaaagaaaac cttagtatta ttaatgagtt 10681 taccatagaa ttgttggaaa tactgaagac aggtgcaatt tactaaactt ttgtttttaa 10741 actattgtag aggctgcatt agaagaaaat gtttataatg acagagcaac tatgactata 10801 taaaaaagct gaaattagaa ctgtgtttag aaatagatca gtaacccagt gccaaggatg 10861 ccaagctgcc accatggtct tggctctccc acaacccagt gtttctgggg taagtttcac 10921 agtttctagg ccctggaata gcaggcagtg taagcctttg ataactttag ttcgatgttt 10981 ttcttgtttt tgtttgttgg tttggtgcat atgatagtgg gtgttatgct attttgctct 11041 tcccatcaaa ataaagaaac ttccagaggt ttactgttaa aaatactgat atttccataa 11101 acgggtttac caagggtgta gtatttcata ccgcctgaaa tgatcagcat tggcacaaat 11161 caaaattcag ccgcctttga aatgcaaaaa tacctttgac tagtaagtac atcctaggag 11221 tttgaaaact taactaaggt ttaaaattta ccttgtttaa agaacttctg acttttgagg 11281 aaaatctagc tttccaagta actaaaatgt acatgagata aacctctcac cactatgtgt 11341 cccttgagaa atgcaacact tttttagtct tcatacttgt aatctataaa agaaattctg 11401 aagtttagac caagttgccc atttctgcgt aattgacata agttctgtta aaaatattat 11461 aagtaattcg tttcggtttg tagatgtttc ccctgacttg ttaaagagga aaccaggaac 11521 tcagtcatgt ttttgtcctg gataatctac ctgttatgcc agtactccca tccgaggggc 11581 atgcccttag ttgcccagat ggagatgcag ttcagtagat ttggggcaaa gtggctacag 11641 ctctgtcttc cattcactca acacctgttc atgactgagc caggtgccca ggacacatcc 11701 taaacagtca gcttctatcc tgtgtcctag ttggggagac agagtgccag ccagcaaccc 11761 tcccaggttt gtaggtttta ggggttttca gttttgtttg ggttttttgt tttttgtttt 11821 tgtttctaca tccttccccg actcccaggc ataatgaggc atgtcttact caatgttatg 11881 caatggattt aggcaaaaat tcattcttag tgtcagccac acaatttttt ttaatgcagt 11941 atattcacct gtaaatagtt tgtgtaaaat ttgacaaaaa aagtatattt actatactgt 12001 aaatatatgt gatgatatat tgtattattt tgcttttttg taaagcagtt agttgctgca 12061 catggataac aacaaaaatt tgattattct cgtgttagta ttgttaactt ctttttgcga 12121 ctgcgttaca tcatttaaag aaaatgctgt gtattgtaaa cttaaattgt atatgataac 12181 ttactgtcct ttccatccgg gcctaaactt tggcagttcc tttgtctaca accttgttaa 12241 tactgtaaac agttgtacgc cagcaggaaa aatactgccc aacagacaaa atcgatcatt 12301 gtaggggaaa atcatagaaa tccatttcag atctttattg ttcctcaccc cattttcctc 12361 cttgtgtatg tacttccccc accccccttt ttttaagtaa aatgtaaatt caatctgctc 12421 taagaaaaaa aaaaaaaaaa aaaa

By "Neurofibromin 1 (NF1) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_001035957, version NP OO 1035957.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 8):

1 maahrpvewv qa vsrfdeq lpiktgqqnt htkvstehnk ecliniskyk fslvisgltt

61 ilknvnnmri fgeaaeknly lsqliildtl ekclagqpkd tmrldetmlv kqllpeichf 121 lhtcregnqh aaelrnsasg vlfslscnnf navfsristr lqeltvcsed nvdvhdiell 181 qyinvdcakl krllketafk fkalkkvaql avinslekaf wnwvenypde ftklyqipqt 241 dmaecaeklf dlvdgfaest krkaavwplq iillilcpei iqdiskd vd ennmnkklf1 301 dslrkalagh ggsrqltesa aiacvklcka styinwedns vifllvqsmv vdlknllfnp 361 skpfsrgsqp advdlmidcl vscfrisphn nqhfkiclaq nspstfhyvl vnslhriitn 421 saldwwpkid avychsvelr nmfgetlhka vqgcgahpai rmapsltfke kvtslkfkek 481 ptdletrsyk ylllsmvkli hadpklllcn prkqgpetqg staelitglv qlvpqshmpe 541 iaqeameall vlhqldsidl wnpdapvetf weissqmlfy ickkltshqm lssteilkwl 601 reilicrnkf llknkqadrs schfllfygv gcdipssgnt sqmsmdheel lrtpgaslrk 661 gkgnssmdsa agcsgtppic rqaqtkleva lymflwnpdt eavlvamscf rhlceeadir 721 cgvdevsvhn llpnyntfme fasvsnmmst graalqkrvm allrriehpt agnteawedt 781 hakweqatkl ilnypkakme dgqaaeslhk tivkrrmshv sgggsidlsd tdslqewinm 841 tgflcalggv clqqrsnsgl atysppmgpv serkgsmisv mssegnadtp vskfmdrlls 901 lmvcnhekvg lqirtnvkdl vglelspaly pmlfnklknt iskffdsqgq vlltdtntqf 961 veqtiaimkn lldnhtegss ehlgqasiet mmlnlvryvr vlgnmvhaiq iktklcqlve 1021 vmmarrddls fcqemkfrnk mveyltdwvm gtsnqaaddd vkcltrdldq asmeavvsll 1081 aglplqpeeg dgvelmeaks qlflkyftlf mnllndcsev edesaqtggr krgmsrrlas 1141 lrhctvlams nllnanvdsg lmhsiglgyh kdlqtratfm evltkilqqg tefdtlaetv 1201 ladrferlve lvtmmgdqge lpiamalanv vpcsqwdela rvlvtlfdsr hllyqllwnm 1261 fskevelads mqtlfrgnsl askimtfcfk vygatylqkl ldpllrivit ssdwqhvsfe 1321 vdptrlepse sleenqrnll qmtekffhai issssefppq lrsvchclyq atchsllnka 1381 tvkekkenkk s vsqrfpqn sigavgsamf lrfinpaivs pyeagildkk ppprierglk 1441 lmskilqsia nhvlftkeeh mrpfndfvks nfdaarrff1 diasdcptsd avnhsls fis 1501 dgnvlalhrl lwnnqekigq ylssnrdhka vgrrpfdkma tllaylgppe hkpvadthws 1561 slnltsskfe efmtrhqvhe keefkalktl sifyqagtsk agnpifyyva rrfktgqing 1621 dlliyhvllt lkpyyakpye ivvdlthtgp snrfktdfIs kwfvvfpgfa ydnvsavyiy 1681 ncnswvreyt kyherlltgl kgskrlvfid cpgklaehie heqqklpaat laleedlkvf 1741 hnalklahkd tkvsikvgst avqvtsaert kvlgqsvfIn diyyaseiee iclvdenqft 1801 ltianqgtpl tfmhqeceai vqsiihirtr welsqpdsip qhtkirpkdv pgtllniall 1861 nlgssdpslr saaynllcal tctfnlkieg qlletsglci panntlfivs isktlaanep 1921 hltlefleec isgfskssie lkhlcleymt pwlsnlvrfc khnddakrqr vtaildklit 1981 mtinekqmyp siqakiwgsl gqitdlldvv Ids fiktsat gglgsikaev madtavalas 2041 gnvklvsskv igrmckiidk tclsptptle qhlmwddiai larymlmls f nnsldvaahl 2101 pylfh vtf1 vatgplslra sthglvinii hslctcsqlh fseetkqvlr lsltefslpk 2161 fyllfgiskv ksaaviafrs syrdrs fspg syeretfalt sletvteall eimeacmrdi 2221 ptckwldqwt elaqrfafqy npslqpralv vfgciskrvs hgqikqiiri lskalesclk 2281 gpdtynsqvl ieatvialtk lqpllnkdsp lhkalfwvav avlqldevnl ysagtalleq 2341 nlhtldslri fndkspeevf mairnplewh ckqmdhfvgl nfnsnfnfal vghllkgyrh 2401 pspaivartv rilhtlltlv nkhrncdkfe vntqsvayla alltvseevr srcslkhrks 2461 llltdismen vpmdtypihh gdpsyrtlke tqpwsspkgs egylaatypt vgqtsprark 2521 smsldmgqps qantkkllgt rks fdhlisd tkapkrqeme sgittppkmr rvaetdyeme 2581 tqrisssqqh phlrkvsvse snvlldeevl tdpkiqalll tvlatlvkyt tdefdqrily 2641 eylaeas vf pkvfpvvhnl ldskintlls lcqdpnllnp ihgivqsvvy heesppqyqt 2701 sylqs fgfng lwrfagpfsk qtqipdyael ivkfldalid tylpgideet seeslltpts 2761 pyppalqsql sitanlnlsn smtslatsqh spgidkenve lspttghcns grtrhgsasq 2821 vqkqrsags f krnsikkiv

By "RB Transcriptional Corepressor 1 (RBI) nucleic acid molecule" is meant a polynucleotide encoding a RBI polypeptide. An exemplary RBI nucleic acid molecule is provided at NCBI Accession No. NM_000321, version NM_000321.2, incorporated herein by reference, and reproduced below (SEQ ID NO: 9):

1 gctcagttgc cgggcggggg agggcgcgtc cggtttttct caggggacgt tgaaattatt 61 tttgtaacgg gagtcgggag aggacggggc gtgccccgac gtgcgcgcgc gtcgtcctcc 121 ccggcgctcc tccacagctc gctggctccc gccgcggaaa ggcgtcatgc cgcccaaaac 181 cccccgaaaa acggccgcca ccgccgccgc tgccgccgcg gaacccccgg caccgccgcc 241 gccgccccct cctgaggagg acccagagca ggacagcggc ccggaggacc tgcctctcgt 301 caggcttgag tttgaagaaa cagaagaacc tgattttact gcattatgtc agaaattaaa 361 gataccagat catgtcagag agagagcttg gttaacttgg gagaaagttt catctgtgga 421 tggagtattg ggaggttata ttcaaaagaa aaaggaactg tggggaatct gtatctttat 481 tgcagcagtt gacctagatg agatgtcgtt cacttttact gagctacaga aaaacataga 541 aatcagtgtc cataaattct ttaacttact aaaagaaatt gataccagta ccaaagttga 601 taatgctatg tcaagactgt tgaagaagta tgatgtattg tttgcactct tcagcaaatt 661 ggaaaggaca tgtgaactta tatatttgac acaacccagc agttcgatat ctactgaaat 721 aaattctgca ttggtgctaa aagtttcttg gatcacattt ttattagcta aaggggaagt 781 attacaaatg gaagatgatc tggtgatttc atttcagtta atgctatgtg tccttgacta 841 ttttattaaa ctctcacctc ccatgttgct caaagaacca tataaaacag ctgttatacc 901 cattaatggt tcacctcgaa cacccaggcg aggtcagaac aggagtgcac ggatagcaaa 961 acaactagaa aatgatacaa gaattattga agttctctgt aaagaacatg aatgtaatat 1021 agatgaggtg aaaaatgttt atttcaaaaa ttttatacct tttatgaatt ctcttggact 1081 tgtaacatct aatggacttc cagaggttga aaatctttct aaacgatacg aagaaattta 1141 tcttaaaaat aaagatctag atgcaagatt atttttggat catgataaaa ctcttcagac 1201 tgattctata gacagttttg aaacacagag aacaccacga aaaagtaacc ttgatgaaga 1261 ggtgaatgta attcctccac acactccagt taggactgtt atgaacacta tccaacaatt 1321 aatgatgatt ttaaattcag caagtgatca accttcagaa aatctgattt cctattttaa 1381 caactgcaca gtgaatccaa aagaaagtat actgaaaaga gtgaaggata taggatacat 1441 ctttaaagag aaatttgcta aagctgtggg acagggttgt gtcgaaattg gatcacagcg 1501 atacaaactt ggagttcgct tgtattaccg agtaatggaa tccatgctta aatcagaaga 1561 agaacgatta tccattcaaa attttagcaa acttctgaat gacaacattt ttcatatgtc 1621 tttattggcg tgcgctcttg aggttgtaat ggccacatat agcagaagta catctcagaa 1681 tcttgattct ggaacagatt tgtctttccc atggattctg aatgtgctta atttaaaagc 1741 ctttgatttt tacaaagtga tcgaaagttt tatcaaagca gaaggcaact tgacaagaga 1801 aatgataaaa catttagaac gatgtgaaca tcgaatcatg gaatcccttg catggctctc 1861 agattcacct ttatttgatc ttattaaaca atcaaaggac cgagaaggac caactgatca 1921 ccttgaatct gcttgtcctc ttaatcttcc tctccagaat aatcacactg cagcagatat 1981 gtatctttct cctgtaagat ctccaaagaa aaaaggttca actacgcgtg taaattctac 2041 tgcaaatgca gagacacaag caacctcagc cttccagacc cagaagccat tgaaatctac 2101 ctctctttca ctgttttata aaaaagtgta tcggctagcc tatctccggc taaatacact 2161 ttgtgaacgc cttctgtctg agcacccaga attagaacat atcatctgga cccttttcca 2221 gcacaccctg cagaatgagt atgaactcat gagagacagg catttggacc aaattatgat 2281 gtgttccatg tatggcatat gcaaagtgaa gaatatagac cttaaattca aaatcattgt 2341 aacagcatac aaggatcttc ctcatgctgt tcaggagaca ttcaaacgtg ttttgatcaa 2401 agaagaggag tatgattcta ttatagtatt ctataactcg gtcttcatgc agagactgaa 2461 aacaaatatt ttgcagtatg cttccaccag gccccctacc ttgtcaccaa tacctcacat 2521 tcctcgaagc ccttacaagt ttcctagttc acccttacgg attcctggag ggaacatcta 2581 tatttcaccc ctgaagagtc catataaaat ttcagaaggt ctgccaacac caacaaaaat 2641 gactccaaga tcaagaatct tagtatcaat tggtgaatca ttcgggactt ctgagaagtt 2701 ccagaaaata aatcagatgg tatgtaacag cgaccgtgtg ctcaaaagaa gtgctgaagg 2761 aagcaaccct cctaaaccac tgaaaaaact acgctttgat attgaaggat cagatgaagc 2821 agatggaagt aaacatctcc caggagagtc caaatttcag cagaaactgg cagaaatgac 2881 ttctactcga acacgaatgc aaaagcagaa aatgaatgat agcatggata cctcaaacaa 2941 ggaagagaaa tgaggatctc aggaccttgg tggacactgt gtacacctct ggattcattg 3001 tctctcacag atgtgactgt ataactttcc caggttctgt ttatggccac atttaatatc 3061 ttcagctctt tttgtggata taaaatgtgc agatgcaatt gtttgggtga ttcctaagcc 3121 acttgaaatg ttagtcattg ttatttatac aagattgaaa atcttgtgta aatcctgcca 3181 tttaaaaagt tgtagcagat tgtttcctct tccaaagtaa aattgctgtg ctttatggat 3241 agtaagaatg gccctagagt gggagtcctg ataacccagg cctgtctgac tactttgcct 3301 tcttttgtag catataggtg atgtttgctc ttgtttttat taatttatat gtatattttt 3361 ttaatttaac atgaacaccc ttagaaaatg tgtcctatct atcttccaaa tgcaatttga 3421 ttgactgccc attcaccaaa attatcctga actcttctgc aaaaatggat attattagaa 3481 attagaaaaa aattactaat tttacacatt agattttatt ttactattgg aatctgatat 3541 actgtgtgct tgttttataa aattttgctt ttaattaaat aaaagctgga agcaaagtat 3601 aaccatatga tactatcata ctactgaaac agatttcata cctcagaatg taaaagaact 3661 tactgattat tttcttcatc caacttatgt ttttaaatga ggattattga tagtactctt 3721 ggtttttata ccattcagat cactgaattt ataaagtacc catctagtac ttgaaaaagt 3781 aaagtgttct gccagatctt aggtatagag gaccctaaca cagtatatcc caagtgcact 3841 ttctaatgtt tctgggtcct gaagaattaa gatacaaatt aattttactc cataaacaga 3901 ctgttaatta taggagcctt aatttttttt tcatagagat ttgtctaatt gcatctcaaa 3961 attattctgc cctccttaat ttgggaaggt ttgtgttttc tctggaatgg tacatgtctt 4021 ccatgtatct tttgaactgg caattgtcta tttatctttt atttttttaa gtcagtatgg 4081 tctaacactg gcatgttcaa agccacatta tttctagtcc aaaattacaa gtaatcaagg 4141 gtcattatgg gttaggcatt aatgtttcta tctgattttg tgcaaaagct tcaaattaaa 4201 acagctgcat tagaaaaaga ggcgcttctc ccctccccta cacctaaagg tgtatttaaa 4261 ctatcttgtg tgattaactt atttagagat gctgtaactt aaaatagggg atatttaagg 4321 tagcttcagc tagcttttag gaaaatcact ttgtctaact cagaattatt tttaaaaaga 4381 aatctggtct tgttagaaaa caaaatttta ttttgtgctc atttaagttt caaacttact 4441 attttgacag ttattttgat aacaatgaca ctagaaaact tgactccatt tcatcattgt 4501 ttctgcatga atatcataca aatcagttag tttttaggtc aagggcttac tatttctggg 4561 tcttttgcta ctaagttcac attagaatta gtgccagaat tttaggaact tcagagatcg 4621 tgtattgaga tttcttaaat aatgcttcag atattattgc tttattgctt ttttgtattg 4681 gttaaaactg tacatttaaa attgctatgt tactattttc tacaattaat agtttgtcta 4741 ttttaaaata aattagttgt taagagtctt aa

By "RB Transcriptional Corepressor 1 (RBI) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.

NP_000312, version NP_000312.2, incorporated herein by reference, as reproduced below (SEQ ID NO: 10):

1 mppktprkta ataaaaaaep papppppppe edpeqdsgpe dlplvrlefe eteepdftal

61 cqklkipdhv rerawltwek vssvdgvlgg yiqkkkelwg icifiaavdl dems ftftel 121 qknieisvhk ffnllkeidt stkvdnamsr llkkydvlfa Ifsklertce liyltqpsss 181 isteinsalv lkvswitf11 akgevlqmed dlvis fqlml cvldyfikls ppmllkepyk 241 tavipingsp rtprrgqnrs ariakqlend triievlcke hecnidevkn vyfknfipfm 301 nslglvtsng lpevenlskr yeeiylknkd ldarlfldhd ktlqtdsids fetqrtprks 361 nldeevnvip phtpvrtvmn tiqqlmmiln sasdqpsenl isyfnnctvn pkesilkrvk 421 digyifkekf akavgqgcve igsqryklgv rlyyrvmesm lkseeerlsi qnfskllndn 481 ifhmsllaca levvmatysr stsqnldsgt dls fpwilnv lnlkafdfyk vies fikaeg 541 nltremikhl ercehrimes lawlsdsplf dlikqskdre gptdhlesac plnlplqnnh 601 taadmylspv rspkkkgstt rvnstanaet qatsafqtqk plkstslslf ykkvyrlayl 661 rlntlcerll sehpelehii wtlfqhtlqn eyelmrdrhl dqimmcsmyg ickvknidlk 721 fkiivtaykd lphavqetfk rvlikeeeyd siivfynsvf mqrlktnilq yastrpptls 781 piphiprspy kfpssplrip ggniyisplk spykiseglp tptkmtprsr ilvsiges fg 841 tsekfqkinq mvcnsdrvlk rsaegsnppk plkklrfdie gsdeadgskh lpgeskfqqk 901 laemtstrtr mqkqkmndsm dtsnkeek

By "C-Src Tyrosine Kinase (CSK) nucleic acid molecule" is meant a polynucleotide encoding a CSK polypeptide. An exemplary CSK nucleic acid molecule is provided at NCBI Accession No. NM_004383, version NM_004383.2, incorporated herein by reference, and reproduced below (SEQ ID NO: 30):

1 ccgggccgcg cttcctctcg ccaggcctgc gagcttcctc ccagcggagc cctgggcgag 61 ccgaggttgg ccgccgccgc cgccgagccc gctgccgccc tcccgctcct gccccacccg 121 cgccttgccc gggggcttct gccggggtgg ggtccgagcc gggcgaccgc ccggctgcgc 181 cgccgtcggg gccgtaaccc ggcccgccgt ccctcccgcc ccagccagcc tctggccgcc 241 ggagcccgcg gggcgtggag cgcgaggagc cccgcggccc cgatcgagcg tccggggcgg 301 cccccggcag ccagcgcgac gttccaaaat cgaacctcag tggcggcgct cggaagcgga 361 actctgccgg ggccgcgccg gctacattgt ttcctccccc cgactccctc ccgccccctt 421 cccccgcctt tcttccctcc gcgacccggg ccgtgcgtcc gtccccctgc ctctgcctgg 481 cggtccctcc tcccctctcc ttgcacccat acctctttgt accgcacccc ctggggaccc 541 ctgcgcccct cccctccccc ctgaccgcat ggaccgtccc gcaggccgct gatgccgccc 601 gcggcgaggt ggcccggacc gcagtgcccc aagagagctc taatggtacc aagtgacagg 661 ttggctttac tgtgactcgg ggacgccaga gctcctgaga agatgtcagc aatacaggcc 721 gcctggccat ccggtacaga atgtattgcc aagtacaact tccacggcac tgccgagcag 781 gacctgccct tctgcaaagg agacgtgctc accattgtgg ccgtcaccaa ggaccccaac 841 tggtacaaag ccaaaaacaa ggtgggccgt gagggcatca tcccagccaa ctacgtccag 901 aagcgggagg gcgtgaaggc gggtaccaaa ctcagcctca tgccttggtt ccacggcaag 961 atcacacggg agcaggctga gcggcttctg tacccgccgg agacaggcct gttcctggtg 1021 cgggagagca ccaactaccc cggagactac acgctgtgcg tgagctgcga cggcaaggtg 1081 gagcactacc gcatcatgta ccatgccagc aagctcagca tcgacgagga ggtgtacttt 1141 gagaacctca tgcagctggt ggagcactac acctcagacg cagatggact ctgtacgcgc 1201 ctcattaaac caaaggtcat ggagggcaca gtggcggccc aggatgagtt ctaccgcagc 1261 ggctgggccc tgaacatgaa ggagctgaag ctgctgcaga ccatcgggaa gggggagttc 1321 ggagacgtga tgctgggcga ttaccgaggg aacaaagtcg ccgtcaagtg cattaagaac 1381 gacgccactg cccaggcctt cctggctgaa gcctcagtca tgacgcaact gcggcatagc 1441 aacctggtgc agctcctggg cgtgatcgtg gaggagaagg gcgggctcta catcgtcact 1501 gagtacatgg ccaaggggag ccttgtggac tacctgcggt ctaggggtcg gtcagtgctg 1561 ggcggagact gtctcctcaa gttctcgcta gatgtctgcg aggccatgga atacctggag 1621 ggcaacaatt tcgtgcatcg agacctggct gcccgcaatg tgctggtgtc tgaggacaac 1681 gtggccaagg tcagcgactt tggtctcacc aaggaggcgt ccagcaccca ggacacgggc 1741 aagctgccag tcaagtggac agcccctgag gccctgagag agaagaaatt ctccactaag 1801 tctgacgtgt ggagtttcgg aatccttctc tgggaaatct actcctttgg gcgagtgcct 1861 tatccaagaa ttcccctgaa ggacgtcgtc cctcgggtgg agaagggcta caagatggat 1921 gcccccgacg gctgcccgcc cgcagtctat gaagtcatga agaactgctg gcacctggac 1981 gccgccatgc ggccctcctt cctacagctc cgagagcagc ttgagcacat caaaacccac 2041 gagctgcacc tgtgacggct ggcctccgcc tgggtcatgg gcctgtgggg actgaacctg 2101 gaagatcatg gacctggtgc ccctgctcac tgggcccgag cctgaactga gccccagcgg 2161 gctggcgggc ctttttcctg cgtcccagcc tgcacccctc cggccccgtc tctcttggac 2221 ccacctgtgg ggcctgggga gcccactgag gggccaggga ggaaggaggc cacggagcgg 2281 gaggcagcgc cccaccacgt cgggcttccc tggcctcccg ccactcgcct tcttagagtt 2341 ttattccttt ccttttttga gatttttttt ccgtgtgttt attttttatt atttttcaag 2401 ataaggagaa agaaagtacc cagcaaatgg gcattttaca agaagtacga atcttatttt 2461 tcctgtcctg cccgtgaggg tgggggggac cgggcccctc tctagggacc cctcgcccca 2521 gcctcattcc ccattctgtg tcccatgtcc cgtgtctcct cggtcgcccc gtgtttgcgc 2581 ttgaccatgt tgcactgttt gcatgcgccc gaggcagacg tctgtcaggg gcttggattt 2641 cgtgtgccgc tgccacccgc ccacccgcct tgtgagatgg aattgtaata aaccacgcca 2701 tgaggacacc gccgcccgcc tcggcgcttc ctccaccgag aaaaaaaaaa aaaaa

By "C-Src Tyrosine Kinase (CSK) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. BAG70102, version BAG70102.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 31):

1 msaiqaawps gteciakynf hgtaeqdlpf ckgdvltiva vtkdpnwyka knkvgregii 61 panyvqkreg vkagtklslm pwfhgkitre qaerllyppe tglflvrest nypgdytlcv 121 gcdgkvehyr imyhasklsi deevyfenlm qlvehytsda dglctrlikp kvmegtvaaq 181 defyrsgwal nmkelkllqt igkgefgdvm lgdyrgnkva vkcikndata qaflaeasvm 241 tqlrhsnlvq llgviveekg glyivteyma kgslvdylrs rgrsvlggdc llkfsldvce 301 ameylegnnf vhrdlaarnv lvsednvakv sdfgltkeas stqdtgklpv kwtapealre 361 kkfstksdvw sfgillweiy sfgrvpypri plkd vprve kgykmdapdg cppavyevmk 421 ncwhldaamr psflqlreql ehikthelhl By "Mitogen-Activated Protein Kinase 8 (MAPK8) nucleic acid molecule" is meant a polynucleotide encoding a MAPK8 polypeptide. An exemplary MAPK8 nucleic acid molecule is provided at NCBI Accession No. NM_001323320, version NM_001323320.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 32):

1 gacgtgcgcg ggcgtgcgcg gtgacggccc gcgtctctgt tactcagccg agcggccgag

61 gccggacgac gcggcttgga ttgcggagcc gcgagcagcg ctgggtaacg gccgcggcga 121 ccaccccgga cggcccctgt ccccgctggc gggcttccct gtcgccgttc gctgcgctgc 181 cggcttcttg gtgaattttt ggatgaagcc attaaattaa ttgcttgcca tcatgagcag 241 aagcaagcgt gacaacaatt tttatagtgt agagattgga gattctacat tcacagtcct 301 gaaacgatat cagaatttaa aacctatagg ctcaggagct caaggaatag tatgcgcagc 361 ttatgatgcc attcttgaaa gaaatgttgc aatcaagaag ctaagccgac catttcagaa 421 tcagactcat gccaagcggg cctacagaga gctagttctt atgaaatgtg ttaatcacaa 481 aaatataatt ggccttttga atgttttcac accacagaaa tccctagaag aatttcaaga 541 tgtttacata gtcatggagc tcatggatgc aaatctttgc caagtgattc agatggagct 601 agatcatgaa agaatgtcct accttctcta tcagatgctg tgtggaatca agcaccttca 661 ttctgctgga attattcatc gggacttaaa gcccagtaat atagtagtaa aatctgattg 721 cactttgaag attcttgact tcggtctggc caggactgca ggaacgagtt ttatgatgac 781 gccttatgta gtgactcgct actacagagc acccgaggtc atccttggca tgggctacaa 841 ggaaaacgct gactcagaac acaacaaact taaagccagt caggcaaggg atttgttatc 901 caaaatgctg gtaatagatg catctaaaag gatctctgta gatgaagctc tccaacaccc 961 gtacatcaat gtctggtatg atccttctga agcagaagct ccaccaccaa agatccctga 1021 caagcagtta gatgaaaggg aacacacaat agaagagtgg aaagaattga tatataagga 1081 agttatggac ttggaggaga gaaccaagaa tggagttata cgggggcagc cctctccttt 1141 aggtgcagca gtgatcaatg gctctcagca tccatcatca tcgtcgtctg tcaatgatgt 1201 gtcttcaatg tcaacagatc cgactttggc ctctgataca gacagcagtc tagaagcagc 1261 agctgggcct ctgggctgct gtagatgact acttgggcca tcggggggtg ggagggatgg 1321 ggagtcggtt agtcattgat agaactactt tgaaaacaat tcagtggtct tatttttggg 1381 tgatttttca aaaaatgtag aattcatttt gtagtaaagt agtttatttt ttttaatttc 1441 aagtgatgta atttaaaacc taagttgtgt ttcaaaacag caacaaaact gtattgtatt 1501 ttttttgctg taattaactg tataatgtaa acctaattat tttatcatgg tttaaatttt 1561 ttgcatattt gctttatctt atgctgctga tttttttaac tgaatttgta agattttgtt 1621 tatcaaagca actattatgt ggtgacttgc ctatatcatg aattatttaa gatttttata 1681 gtttttttta attagaattt atttcagatg ttttgttcat gatactatcc ttcagggtta 1741 tgtgcttatc aatgaaataa ccccagagga gtgagggaaa ataacttgta gccagttata 1801 ttcaggaata actactgtaa atgatgaacg tgttaggaga cctccaatat ttgctacttg 1861 ccaatcctaa tttagttaca agaattggta ggcaatccta cttaattttg gcaaaagccc 1921 cgtcatctaa atggcagaat aactcagagc atgtctttga agatgctggg cgtctaccac 1981 caccttatgt ccccacccta cccaacaaaa ataagtaaaa agaatatggt gtattctaca 2041 aatttgtggc atgctcaaag tttatgatca cataaaggca agaggatact tcatgaataa 2101 tacatttcaa tgcaaataaa cagatggttc acttctacta gctatgagcc tgtttttgta 2161 tacactgagt taatctactc aggctgtagg tcccagcaat gttctagagt ctggtctttc 2221 cctttcctgc agcttcgggt ccttggacct ttcctgtttc ctattacttg gagtgtctgt 2281 cagttgagca ccagttgttc tggtgtttca tttgattcta cttgtagcat aatcatttat 2341 acgagctatt gggaggttcc aaaccctacc tagatttgtg taggtgatgt atcaaatgag 2401 caatataccg ttcatctgaa aatagtagca cacagccata tataggatat cattttctaa 2461 ggactgtttc ttcacattga gcagagcagg cataaatggt ggttatttag tctaagtctt 2521 ttattttttt atacctgatt ttcaacataa cacgcaatgt ggatgtcgag tagtgttaag 2581 aatggtgctg ctcctgacaa gtgtatgtta actgtttaca ttttctatct gtagaattat 2641 ttctctatta ctgaactttt cctaagtaaa atgtctttga agtctcgtta tttctgaaat 2701 acgttgtctg taatagaccc aggcaccttt taaattatct ctggaacaag agggatttca 2761 tgtaatgaac taggaaatgc atactcacat aagcaacaag gttctaggca gaaagcccct 2821 tggaatttgt gaccaacagg agcaagaaca ggtgcggctc aacatgcaat gtctgaaaat 2881 ttgcttggca ttttattcat atatttagtg caaaattatt tttgagtgag atattttaca 2941 tcactgttaa tgtgcaatat ttaagattaa aatacattag cttttttata tactttgaag 3001 tagcaagttt gttttcgatg gcttagagtc atgatttcca gcttcccagc ctttttatca 3061 gtcccttttc taatacaaca aggtgcatta atttgattag gcaaattaga gttctaagac 3121 acttcttgaa ttgtagacag aaaatattgg attcacaatt tcagcagaaa tttgagaatg 3181 agtgtgttta tattaatttc acaattagct gtattttctg tagcatagat tatgtcactg 3241 ttgcactttc acagcagaca tgctttcaga aggttctcat attttatgtt tgattgctga 3301 taagccatct ctattgatac agattttggt taagtaagga aaaccaggtg tgtgtctgta 3361 tcatttattg taaatgccag ctgccacttg ccaaccatca tgttcagttc aattcaaaga 3421 aaacaaactc tcattactta gtgtaaacta aaatacttaa caaattatat cctaaaaaca 3481 aggtctcttt gttaaatgtt gcatgcccta ggttttaaat tactacatcc aaatacagtt 3541 ttcgtcttaa atttgttaag ctaaatatat gttggttctt tttattttgg aatcctttaa 3601 gcatcttaaa catttttttt ttgaagagaa gttacaaata acatttctat caggtagtac 3661 ttgtatgaaa ccacctttct tattctataa ttttgatttt tcaattttat atacttaata 3721 tactcactgt cttactatca gaaagttatt ttgaccaaga tttttattat cttcatagat 3781 tcagaaagag atgctaattc tgtaccaatg tcttcctggt tactattctc ttccctctaa 3841 tatatactgg ccatttgtaa aaccattgtg ttgttgggat cacttagtta tactatacgc 3901 agatagagca tctcaactct gtcatagtgt ttgctgaaca gttttcagtg tcatgcacct 3961 ttggctgcta attgttcctg acgtgcactc ttccgagttg gtaaaggcac agtgtgttca 4021 tgccagactt ctaagagaaa caccagcctc ttaaatcaga agcctacaca caaccccctt 4081 aacaatccaa agaagcttga tggtgtgcaa agaagcatcc tgccagcctt gtcattgttc 4141 tgttctatgc taatcctgct gtgttgtcta aaagatggag ggaagaggac atcagtgtct 4201 gatagtgaaa tcatcagcag gaaagtgaag ctctttcctt ggttacagat aagacttggt 4261 ttacactatt ggccagtatc tgctaaacat atgaagactt aactattcag tgttgcctag 4321 gcattcgcct gcacaacatt ttgaggttag aacatagaat attttcagaa atactgttgt 4381 agtttgtgag tgttgttcat tagttacaca ttagctatag agtggatgca tgaagcccca 4441 tgacaccagt aaacttctct taccagtagg taaaccaaac accattctgt cattagcagc 4501 cctcttaaat gttgcctctc cgtatcctgt tgcatttttg tgtgcattgt gtttctactg 4561 atctctctta ggtttttacg gaatcaaagg aaactaattt ttccttaata gcaagaaaga 4621 tgaagaggta aagggcattg aagcagaaat gtatagtttg gggtacgatt agaaaactcg 4681 taaggaaaac agaagtccta atttcaaact gactgctctt cgttaagtgc tcttaaggag 4741 agtctagtaa cagtaacact ttctggccat ttctagttta gattctcttc gttactgaaa 4801 cttttgagaa atattacctg tggattaatt ttgcacaatg ttctattctc ataatgactt 4861 acaaattaaa ctaggttttt attgaactac ctcacactaa ttttctatgc tttcccaagt 4921 aagctgttgc cctgttagat ctttactgag tgaattataa atgtgtgtta aatactttct 4981 agccaatgtt gacacaatac cagtaagtat gtaaagtata taccttacat cagtaagaga 5041 cacgtgtaaa atctttgact gtatgtcttg caaaattgtg ctcgttgaca ttattactgt 5101 ttttgtaagt agaaacctgc tcgtgatatc ggtccattta cattttacaa aaggagtaaa 5161 tcttagtaaa aattttacga agaaataaat tacttttgta ggcccaatat ttggtatatt 5221 tttgagaagc tgttaatctt ttagctgaat aatgaagtta gactgaatta cgtgtctccc 5281 tggactgtga catctatttt ctcattacag tttatcctgg tcagcagggt gtcacacctg 5341 gaaacctgag tatgatagct gacatttgct tttctccctc tgcgatgtca ttcctcctcc 5401 attcctctcc ttccctgtgt tccgttccct ctcctttcct ctagacaaaa caaaatgggg 5461 cactttttag ggaatgctga gatcattatt gtggtttttc atcattcatg ccctagtcat 5521 taaacatgca ccactggaat gtaaacaatg ttatctagta tgtcaattgg ttataatatt 5581 ttaaataaaa aagaaaaaag tggtatgaaa attatgaaa

By "Mitogen-Activated Protein Kinase 8 (MAPK8)" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAI30573, version AAI30573.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 33): 1 msrskrdnnf ysveigdstf tvlkryqnlk pigsgaqgiv caaydailer nvaikklsrp

61 fqnqthakra yrelvlmkcv nhkniiglln vftpqkslee fqdvyivmel mdanlcqviq

121 meldhermsy llyqmlcgik hlhsagiihr dlkpsni vk sdctlkildf glartagts f

181 mmtpy vtry yrapevilgm gykenvdiws vgcimgemik ggvl fpgtdh idqwnkvieq

241 lgtpcpefmk klqptvrtyv enrpkyagys feklfpdvlf padsehnklk asqardllsk

301 mlvidaskri svdealqhpy invwydpsea eapppkipdk qlderehtie ewkeliykev

361 mdleertkng virgqpsplg aavingsqhp sssssvndvs smstdptlas dtdssleaaa

421 gplgccr

By "Janus Kinase 3 (JAK3) nucleic acid molecule" is meant a polynucleotide encoding a JAK3 polypeptide. An exemplary JAK3 nucleic acid molecule is provided at NCBI Accession No. NM_000215, version NM_000215.3, incorporated herein by reference, and reproduced below (SEQ ID NO: 34):

1 cacacaggaa ggagccgagt gggactttcc tctcgctgcc tcccggctct gcccgccctt

61 cgaaagtcca gggtccctgc ccgctaggca agttgcactc atggcacctc caagtgaaga 121 gacgcccctg atccctcagc gttcatgcag cctcttgtcc acggaggctg gtgccctgca 181 tgtgctgctg cccgctcggg gccccgggcc cccccagcgc ctatctttct cctttgggga 241 ccacttggct gaggacctgt gcgtgcaggc tgccaaggcc agcggcatcc tgcctgtgta 301 ccactccctc tttgctctgg ccacggagga cctgtcctgc tggttccccc cgagccacat 361 cttctccgtg gaggatgcca gcacccaagt cctgctgtac aggattcgct tttacttccc 421 caattggttt gggctggaga agtgccaccg cttcgggcta cgcaaggatt tggccagtgc 481 tatccttgac ctgccagtcc tggagcacct ctttgcccag caccgcagtg acctggtgag 541 tgggcgcctc cccgtgggcc tcagtctcaa ggagcagggt gagtgtctca gcctggccgt 601 gttggacctg gcccggatgg cgcgagagca ggcccagcgg ccgggagagc tgctgaagac 661 tgtcagctac aaggcctgcc tacccccaag cctgcgcgac ctgatccagg gcctgagctt 721 cgtgacgcgg aggcgtattc ggaggacggt gcgcagagcc ctgcgccgcg tggccgcctg 781 ccaggcagac cggcactcgc tcatggccaa gtacatcatg gacctggagc ggctggatcc 841 agccggggcc gccgagacct tccacgtggg cctccctggg gcccttggtg gccacgacgg 901 gctggggctg ctccgcgtgg ctggtgacgg cggcatcgcc tggacccagg gagaacagga 961 ggtcctccag cccttctgcg actttccaga aatcgtagac attagcatca agcaggcccc 1021 gcgcgttggc ccggccggag agcaccgcct ggtcactgtt accaggacag acaaccagat 1081 tttagaggcc gagttcccag ggctgcccga ggctctgtcg ttcgtggcgc tcgtggacgg 1141 ctacttccgg ctgaccacgg actcccagca cttcttctgc aaggaggtgg caccgccgag 1201 gctgctggag gaagtggccg agcagtgcca cggccccatc actctggact ttgccatcaa 1261 caagctcaag actgggggct cacgtcctgg ctcctatgtt ctccgccgca gcccccagga 1321 ctttgacagc ttcctcctca ctgtctgtgt ccagaacccc cttggtcctg attataaggg 1381 ctgcctcatc cggcgcagcc ccacaggaac cttccttctg gttggcctca gccgacccca 1441 cagcagtctt cgagagctcc tggcaacctg ctgggatggg gggctgcacg tagatggggt 1501 ggcagtgacc ctcacttcct gctgtatccc cagacccaaa gaaaagtcca acctgatcgt 1561 ggtccagaga ggtcacagcc cacccacatc atccttggtt cagccccaat cccaatacca 1621 gctgagtcag atgacatttc acaagatccc tgctgacagc ctggagtggc atgagaacct 1681 gggccatggg tccttcacca agatttaccg gggctgtcgc catgaggtgg tggatgggga 1741 ggcccgaaag acagaggtgc tgctgaaggt catggatgcc aagcacaaga actgcatgga 1801 gtcattcctg gaagcagcga gcttgatgag ccaagtgtcg taccggcatc tcgtgctgct 1861 ccacggcgtg tgcatggctg gagacagcac catggtgcag gaatttgtac acctgggggc 1921 catagacatg tatctgcgaa aacgtggcca cctggtgcca gccagctgga agctgcaggt 1981 ggtcaaacag ctggcctacg ccctcaacta tctggaggac aaaggcctgc cccatggcaa 2041 tgtctctgcc cggaaggtgc tcctggctcg ggagggggct gatgggagcc cgcccttcat 2101 caagctgagt gaccctgggg tcagccccgc tgtgttaagc ctggagatgc tcaccgacag 2161 gatcccctgg gtggcccccg agtgtctccg ggaggcgcag acacttagct tggaagctga 2221 caagtggggc ttcggcgcca cggtctggga agtgtttagt ggcgtcacca tgcccatcag 2281 tgccctggat cctgctaaga aactccaatt ttatgaggac cggcagcagc tgccggcccc 2341 caagtggaca gagctggccc tgctgattca acagtgcatg gcctatgagc cggtccagag 2401 gccctccttc cgagccgtca ttcgtgacct caatagcctc atctcttcag actatgagct 2461 cctctcagac cccacacctg gtgccctggc acctcgtgat gggctgtgga atggtgccca 2521 gctctatgcc tgccaagacc ccacgatctt cgaggagaga cacctcaagt acatctcaca 2581 gctgggcaag ggcaactttg gcagcgtgga gctgtgccgc tatgacccgc taggcgacaa 2641 tacaggtgcc ctggtggccg tgaaacagct gcagcacagc gggccagacc agcagaggga 2701 ctttcagcgg gagattcaga tcctcaaagc actgcacagt gatttcattg tcaagtatcg 2761 tggtgtcagc tatggcccgg gccgccagag cctgcggctg gtcatggagt acctgcccag 2821 cggctgcttg cgcgacttcc tgcagcggca ccgcgcgcgc ctcgatgcca gccgcctcct 2881 tctctattcc tcgcagatct gcaagggcat ggagtacctg ggctcccgcc gctgcgtgca 2941 ccgcgacctg gccgcccgaa acatcctcgt ggagagcgag gcacacgtca agatcgctga 3001 cttcggccta gctaagctgc tgccgcttga caaagactac tacgtggtcc gcgagccagg 3061 ccagagcccc attttctggt atgcccccga atccctctcg gacaacatct tctctcgcca 3121 gtcagacgtc tggagcttcg gggtcgtcct gtacgagctc ttcacctact gcgacaaaag 3181 ctgcagcccc tcggccgagt tcctgcggat gatgggatgt gagcgggatg tccccgccct 3241 ctgccgcctc ttggaactgc tggaggaggg ccagaggctg ccggcgcctc ctgcctgccc 3301 tgctgaggtt cacgagctca tgaagctgtg ctgggcccct agcccacagg accggccatc 3361 attcagcgcc ctgggccccc agctggacat gctgtggagc ggaagccggg ggtgtgagac 3421 tcatgccttc actgctcacc cagagggcaa acaccactcc ctgtcctttt catagctcct 3481 gcccgcagac ctctggatta ggtctctgtt gactggctgt gtgaccttag gcccggagct 3541 gcccctctct gggcctcaga ggccttatga gggtcctcta cttcaggaac acccccatga 3601 cattgcattt gggggggctc ccgtggcctg tagaatagcc tgtggccttt gcaatttgtt 3661 aaggttcaag acagatgggc atatgtgtca gtggggctct ctgagtcctg gcccaaagaa 3721 gcaaggaacc aaatttaaga ctctcgcatc ttcccaaccc cttaagccct ggccccctga 3781 gtttcctttt ctgtctctct ctttttattt tttttatttt tatttttatt tttgagacag 3841 agcctcgctc tgttacccag ggtggagtgc agtggtgcga tctcggctca gtgcaacctc 3901 tgcttcccag gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattacaggt 3961 gtgcaccacc acacccggct aatttttttt atttttaata gagatgaggt ttcaccatga 4021 tggccaggct gatctcgaac tcctaacctc aagtgatcct cccacctcag cctcccaaag 4081 tgttggaata ataggcatga gccactgcac ccaggctttt ttttttttaa atttattatt 4141 attattttta agagacagga tcttgctacg ttgcccaggc tggtcttgaa ctcctgggct 4201 acagtgatcc tcctgcctta tcctcctaaa tagctgggac tacagcacct agttttgagt 4261 ttcctgtctt atttccaatg gggacattca tgtagctttt tttttttttt tttttttgag 4321 acggagtctc gctctgtcgc ccaggctgga gtacagtggc gcaatctagg ctcactgcaa 4381 gctccgcctc ctgggttcac accattctct cgcctcagcc tcccaagtag ctgggactac 4441 aggcgcccgc caccacaccc ggctaatttt ttgtattttt agtagagacg gggtttcacc 4501 ttgttagcca ggatggtttc catctcctga cctcgtgatc tgcccgtctc ggcctcccaa 4561 agtgctggga ttacaggcat gagccactgc gcccggccct catgtagctt taaatgtatg 4621 atctgacttc tgctccccga tctctgtttc tctggaggaa gccaaggaca agagcagttg 4681 ctgtggctgg gactctgcct tttaggggag cccgtgtatc tctttgggat cctgaaaggg 4741 ggcaggaaag gctggggtcc cagtccaccc taatggtatc tgagtgtcct agggcttcag 4801 ttttcccacc tgtccaatgg gaccctttct gtcctcaccc tacaaggggc acaaagggat 4861 gacaccaaac ctggcaggaa cttttcacgc aatcaaggga aggaaaggca ttcctggcag 4921 agggaacagc atgccaagcg tgagaaggct cagagtaagg aggttaagag cccaagtatt 4981 ggagcctaca gttttgcccc ttccatgcag tgtgacagtg ggcaagttcc tttccctctc 5041 tgggtctcag ttctgtcccc tgcaaaatgg tcagagctta ccccttggct gtgcagggtc 5101 aactttctga ctggtgagag ggattctcat gcaggttaag cttctgctgc tcctcctcac 5161 ctgcaaagct tttctgccac ttttgcctcc ttggaaaact cttatccatc tctcaaaact 5221 ccagctacca catccttgca gccttccctc atataccccc actactactg tagccctgtc 5281 cttccctcca gccccactct ggccctgggg ctggggaagt gtctgtgtcc agctgtctcc 5341 cctgacctca gggttccttg ggggctgggc tgaggcctca gtacagaggg ggctctggaa 5401 atgtttgttg actgaataaa ggaattcagt ggaaaaaaaa aaaaaaaaa By "Janus Kinase 3 (JAK3)" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAC50950, version AAC50950.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 35):

1 mappseetpl ipqrscslls teagalhvll pargpgppqr lsfssgdhla edlcvqaaka

61 sgilpvyhsl falatedlsc wfppshifsv edastqvlly rirs fyfpnw fglekchrfg 121 lrkdlasail dlpvlehlfa qhrsdlvsgr lpvglslkeq geclslavld larmareqaq 181 rpgellktvs ykaclppslr dliqgls f t rrairrtvrr alprvaacqa drhslmakyi 241 mdlerldpag aaetfhvglp galgghdglg Ifrvagdggi awtqgeqvlq pfcdfpeivd 301 isikqaprvg pagehrlvtv trtdnqilea efpglpeals f alvdgyfr lttdsqhffc 361 kevapprlle evaeqchgpi tldfainklk tggsrpgsyv lrrspqdfds flltvcvqnp 421 lgpdykgcli rrsptgtf11 vglsrphssl rellatcwdg glhvdgvavt ltscciprpk 481 eksnli vqr ghspptsslv qpqsqyqlsq mtfhkipads lewhenlghg s ftkiyrgcr 541 he vdgeark tevllkvmda khkncmes f1 eaaslmsqvs yrhlvllhgv cmagdstmvq 601 ef hlgaidm ylrkrghlvp aswklq vkq layalnyled kglphgnvsa rkvllarega 661 dgsppfikls dpgvspavls lemltdripw vapeclreaq tlsleadkwg fgatvwevfs 721 gvtmpisald pakklqfyed rqqlpapkwt elalliqqcm ayepvqrps f ravirdlnsl 781 issdyellsd ptpgalaprd glwngaqlya cqdptifeer hlkyisqlgk gnfgsvelcr 841 ydplgdntga lvavkqlqhs gpdqqrdfqr eiqilkalhs dfivkyrgvs ygpgepelrl 901 vmeylpsgcl rdflqrhrar ldasrlllys sqickgmeyl gsrrcvhrdl aarnilvese 961 ahvkiadfgl akllpldkdy y vrepgqsp ifwyapesls dnifsrqsdv ws fgvvlyel 1021 ftycdkscsp saeflrmmgc erdvpalcrl lelleegqrl pappacpaev helmklcwap 1081 spqdrps fsa lgpqldmlws gsrgcethaf tahpegkhhs lsfs

By "Cyclin Dependent Kinase 12 (CDK12) nucleic acid molecule" is meant a polynucleotide encoding a CDK12 polypeptide. An exemplary CDK12 nucleic acid molecule is provided at NCBI Accession No. NM_015083, version NM_015083.2, incorporated herein by reference, and reproduced below (SEQ ID NO: 36):

1 gtgtgactgg gtctgtgtga gggagagagt gtgtgtggtg tggaggtgaa acggaggcaa

61 gaaagggggc tacctcagga gcgagggaca aagggggcgt gaggcaccta ggccgcggca 121 ccccggcgac aggaagccgt cctgaaccgg gctaccgggt aggggaaggg cccgcgtagt 181 cctcgcaggg ccccagagct ggagtcggct ccacagcccc gggccgtcgg cttctcactt 241 cctggacctc cccggcgccc gggcctgagg actggctcgg cggagggaga agaggaaaca 301 gacttgagca gctccccgtt gtctcgcaac tccactgccg aggaactctc atttcttccc 361 tcgctccttc accccccacc tcatgtagaa gggtgctgag gcgtcgggag ggaggaggag 421 cctgggctac cgtccctgcc ctccccaccc ccttcccggg gcgctttggt gggcgtggag 481 ttggggttgg gggggtgggt gggggttgct ttttggagtg ctggggaact tttttccctt 541 cttcaggtca ggggaaaggg aatgcccaat tcagagagac atgggggcaa gaaggacggg 601 agtggaggag cttctggaac tttgcagccg tcatcgggag gcggcagctc taacagcaga 661 gagcgtcacc gcttggtatc gaagcacaag cggcataagt ccaaacactc caaagacatg 721 gggttggtga cccccgaagc agcatccctg ggcacagtta tcaaaccttt ggtggagtat 781 gatgatatca gctctgattc cgacaccttc tccgatgaca tggccttcaa actagaccga 841 agggagaacg acgaacgtcg tggatcagat cggagcgacc gcctgcacaa acatcgtcac 901 caccagcaca ggcgttcccg ggacttacta aaagctaaac agaccgaaaa agaaaaaagc 961 caagaagtct ccagcaagtc gggatcgatg aaggaccgga tatcgggaag ttcaaagcgt 1021 tcgaatgagg agactgatga ctatgggaag gcgcaggtag ccaaaagcag cagcaaggaa 1081 tccaggtcat ccaagctcca caaggagaag accaggaaag aacgggagct gaagtctggg 1141 cacaaagacc ggagtaaaag tcatcgaaaa agggaaacac ccaaaagtta caaaacagtg 1201 gacagcccaa aacggagatc caggagcccc cacaggaagt ggtctgacag ctccaaacaa 1261 gatgatagcc cctcgggagc ttcttatggc caagattatg accttagtcc ctcacgatct 1321 catacctcga gcaattatga ctcctacaag aaaagtcctg gaagtacctc gagaaggcag 1381 tcggtcagtc ccccttacaa ggagccttcg gcctaccagt ccagcacccg gtcaccgagc 1441 ccctacagta ggcgacagag atctgtcagt ccctatagca ggagacggtc gtccagctac 1501 gaaagaagtg gctcttacag cgggcgatcg cccagtccct atggtcgaag gcggtccagc 1561 agccctttcc tgagcaagcg gtctctgagt cggagtccac tccccagtag gaaatccatg 1621 aagtccagaa gtagaagtcc tgcatattca agacattcat cttctcatag taaaaagaag 1681 agatccagtt cacgcagtcg tcattccagt atctcacctg tcaggcttcc acttaattcc 1741 agtctgggag ctgaactcag taggaaaaag aaggaaagag cagctgctgc tgctgcagca 1801 aagatggatg gaaaggagtc caagggttca cctgtatttt tgcctagaaa agagaacagt 1861 tcagtagagg ctaaggattc aggtttggag tctaaaaagt tacccagaag tgtaaaattg 1921 gaaaaatctg ccccagatac tgaactggtg aatgtaacac atctaaacac agaggtaaaa 1981 aattcttcag atacagggaa agtaaagttg gatgagaact ccgagaagca tcttgttaaa 2041 gatttgaaag cacagggaac aagagactct aaacccatag cactgaaaga ggagattgtt 2101 actccaaagg agacagaaac atcagaaaag gagacccctc cacctcttcc cacaattgct 2161 tctcccccac cccctctacc aactactacc cctccacctc agacaccccc tttgccacct 2221 ttgcctccaa taccagctct tccacagcaa ccacctctgc ctccttctca gccagcattt 2281 agtcaggttc ctgcttccag tacttcaact ttgccccctt ctactcactc aaagacatct 2341 gctgtgtcct ctcaggcaaa ttctcagccc cctgtacagg tttctgtgaa gactcaagta 2401 tctgtaacag ctgctattcc acacctgaaa acttcaacgt tgcctccttt gcccctccca 2461 cccttattac ctggagatga tgacatggat agtccaaaag aaactcttcc ttcaaaacct 2521 gtgaagaaag agaaggaaca gaggacacgt cacttactca cagaccttcc tctccctcca 2581 gagctccctg gtggagatct gtctccccca gactctccag aaccaaaggc aatcacacca 2641 cctcagcaac catataaaaa gagaccaaaa atttgttgtc ctcgttatgg agaaagaaga 2701 caaacagaaa gcgactgggg gaaacgctgt gtggacaagt ttgacattat tgggattatt 2761 ggagaaggaa cctatggcca agtatataaa gccaaggaca aagacacagg agaactagtg 2821 gctctgaaga aggtgagact agacaatgag aaagagggct tcccaatcac agccattcgt 2881 gaaatcaaaa tccttcgtca gttaatccac cgaagtgttg ttaacatgaa ggaaattgtc 2941 acagataaac aagatgcact ggatttcaag aaggacaaag gtgcctttta ccttgtattt 3001 gagtatatgg accatgactt aatgggactg ctagaatctg gtttggtgca cttttctgag 3061 gaccatatca agtcgttcat gaaacagcta atggaaggat tggaatactg tcacaaaaag 3121 aatttcctgc atcgggatat taagtgttct aacattttgc tgaataacag tgggcaaatc 3181 aaactagcag attttggact tgctcggctc tataactctg aagagagtcg cccttacaca 3241 aacaaagtca ttactttgtg gtaccgacct ccagaactac tgctaggaga ggaacgttac 3301 acaccagcca tagatgtttg gagctgtgga tgtattcttg gggaactatt cacaaagaag 3361 cctatttttc aagccaatct ggaactggct cagctagaac tgatcagccg actttgtggt 3421 agcccttgtc cagctgtgtg gcctgatgtt atcaaactgc cctacttcaa caccatgaaa 3481 ccgaagaagc aatatcgaag gcgtctacga gaagaattct ctttcattcc ttctgcagca 3541 cttgatttat tggaccacat gctgacacta gatcctagta agcggtgcac agctgaacag 3601 accctacaga gcgacttcct taaagatgtc gaactcagca aaatggctcc tccagacctc 3661 ccccactggc aggattgcca tgagttgtgg agtaagaaac ggcgacgtca gcgacaaagt 3721 ggtgttgtag tcgaagagcc acctccatcc aaaacttctc gaaaagaaac tacctcaggg 3781 acaagtactg agcctgtgaa gaacagcagc ccagcaccac ctcagcctgc tcctggcaag 3841 gtggagtctg gggctgggga tgcaataggc cttgctgaca tcacacaaca gctgaatcaa 3901 agtgaattgg cagtgttatt aaacctgctg cagagccaaa ccgacctgag catccctcaa 3961 atggcacagc tgcttaacat ccactccaac ccagagatgc agcagcagct ggaagccctg 4021 aaccaatcca tcagtgccct gacggaagct acttcccagc agcaggactc agagaccatg 4081 gccccagagg agtctttgaa ggaagcaccc tctgccccag tgatcctgcc ttcagcagaa 4141 cagacgaccc ttgaagcttc aagcacacca gctgacatgc agaatatatt ggcagttctc 4201 ttgagtcagc tgatgaaaac ccaagagcca gcaggcagtc tggaggaaaa caacagtgac 4261 aagaacagtg ggccacaggg gccccgaaga actcccacaa tgccacagga ggaggcagca 4321 gagaagaggc cccctgagcc ccccggacct ccaccgccgc cacctccacc ccctctggtt 4381 gaaggcgatc tttccagcgc cccccaggag ttgaacccag ccgtgacagc cgccttgctg 4441 caacttttat cccagcctga agcagagcct cctggccacc tgccacatga gcaccaggcc 4501 ttgagaccaa tggagtactc cacccgaccc cgtccaaaca ggacttatgg aaacactgat 4561 gggcctgaaa cagggttcag tgccattgac actgatgaac gaaactctgg tccagccttg 4621 acagaatcct tggtccagac cctggtgaag aacaggacct tctcaggctc tctgagccac 4681 cttggggagt ccagcagtta ccagggcaca gggtcagtgc agtttccagg ggaccaggac 4741 ctccgttttg ccagggtccc cttagcgtta cacccggtgg tcgggcaacc attcctgaag 4801 gctgagggaa gcagcaattc tgtggtacat gcagagacca aattgcaaaa ctatggggag 4861 ctggggccag gaaccactgg ggccagcagc tcaggagcag gccttcactg ggggggccca 4921 actcagtctt ctgcttatgg aaaactctat cgggggccta caagagtccc accaagaggg 4981 ggaagaggga gaggagttcc ttactaaccc agagacttca gtgtcctgaa agattccttt 5041 cctatccatc cttccatcca gttctctgaa tctttaatga aatcatttgc cagagcgagg 5101 taatcatctg catttggcta ctgcaaagct gtccgttgta ttccttgctc acttgctact 5161 agcaggcgac ttacgaaata atgatgttgg caccagttcc ccctggatgg gctatagcca 5221 gaacatttac ttcaactcta ccttagtaga tacaagtaga gaatatggag aggatcatta 5281 cattgaaaag taaatgtttt attagttcat tgcctgcact tactgatcgg aagagagaaa 5341 gaacagtttc agtattgaga tggctcagga gaggctcttt gatttttaaa gttttggggt 5401 gggggattgt gtgtggtttc tttcttttga attttaattt aggtgttttg ggtttttttc 5461 ctttaaagag aatagtgttc acaaaatttg agctgctctt tggcttttgc tataagggaa 5521 acagagtggc ctggctgatt tgaataaatg tttctttcct ctccaccatc tcacattttg 5581 cttttaagtg aacacttttt ccccattgag catcttgaac atactttttt tccaaataaa 5641 ttactcatcc ttaaagttta ctccactttg acaaaagata cgcccttctc cctgcacata 5701 aagcaggttg tagaacgtgg cattcttggg caagtaggta gactttaccc agtctctttc 5761 cttttttgct gatgtgtgct ctctctctct ctttctctct ctctctctct ctctctctct 5821 ctctctctct ctctgtctcg cttgctcgct ctcgctgttt ctctctcttt gaggcatttg 5881 tttggaaaaa atcgttgaga tgcccaagaa cctgggataa ttctttactt tttttgaaat 5941 aaaggaaagg aaattcagac tcttacattg ttctctgtaa ctcttcaatt ctaaaatgtt 6001 ttgtttttta aaccatgttc tgatggggaa gttgatttgt aagtgtggac agcttggaca 6061 ttgctgctga gctgtggtta gagatgatgc ctccattcct agagggctaa taacagcatt 6121 tagcatattg tttacacata tatttttatg tcaaaaaaaa aacaaaaacc tttcaaacag 6181 agcattgtga tattgtcaaa gagaaaaaca aatcctgaag atacatggaa atgtaaccta 6241 gtttagggtg ggtatttttc tgaagataca tcaatacctg acctttttta aaaaaataat 6301 tttaaaacag catactgtga ggaagaacag tattgacata cccacatccc agcatgtgta 6361 ccctgccagt tcttttaggg atttttcctc caaagagatt tggatttggt tttggtaaaa 6421 ggggttaaat tgtgcttcca ggcaagaact ttgccttatc ataaacagga aatgaaaaag 6481 ggaagggctg tcaggatggg ataatttggg aggcttctca ttctggcttc tatttctatg 6541 tgagtaccag catatagagt gttttaaaaa cagatacatg tcatataatt tatctgcaca 6601 gacttagacc ttcaggaaac ataggttaag ccccctttta caaagaaaaa gtaaacatac 6661 ttcagcatct tggagggtag ttttcaaaac tcaagtttca tgtttcaatg ccaagttctt 6721 attttaaaaa ataaaatcta cttataagag aaaggtgcat tacttaaaaa aaaaaaactt 6781 taaagaaatg aaagaagaac cctcttcaga tacttacttg aagactgttt tcccctgtta 6841 atgagatata gctagatatc ggtgtgtgta tttctttatt attctctggt ttttgatctg 6901 gccttgcctc cagggccaaa cactgattta gaaagagagc cttctagcta ttttggcatt 6961 gatggctttt tataccagtg tgtccagtta gatttactag gcttactgac atgctattgg 7021 taaatcgcat taaagttcat ctgaaccttc tgtctgttga cttcttagtc ctcagacatg 7081 ggcctttgtg ttttagaata tttgaatttg agttattggg ccccactccc tgttttttat 7141 taaagaacgt gagcctggga tactttcaga agtatctgtt caatgaaaaa aagttggttt 7201 cccatcaaat atgaataaaa ttctctatat atttcattgt attttggtta tcagcagtca 7261 tcaataatgt ttttccctcc cctctcccac ctcttatttt taattatgcc aaatatccta 7321 aataatatac ttaagcctcc attccctcat ccctactagg gaagggggtg agtgtatgtg 7381 tgagtgtatg tgtatgtatg atcccatctc acccccaccc ccattttggg agtcttttaa 7441 aatgaaaaca aagtttggta gttttgacta tttctaaaag cagaggagaa aaaaaaactt 7501 atttaaatat cctggaatct gtatggagga agaaaaggta tttgttaatt tttcagttac 7561 gttatctata aacatgatgg aagtaaaggt ttggcagaat ttcaccttga ctatttgaaa 7621 attacagacc caattaattc cattcaaaag tggttttcgt tttgttttaa ttattgtaca 7681 atgagagata ttgtctatta aatacattat tttgaacaga tgagaaatct gattctgttc

7741 atgagtggga ggcaaaactg gtttgaccgt gatcattttt gtggttttga aaacaaatat

7801 acttgaccca gtttccttag ttttttcttc aactgtccat aggaacgata agtatttgaa

7861 agcaacatca aatctatacg tttaaagcag ggcagttagc acaaatttgc aagtagaact

7921 tctattagct tatgccatag acatcaccca accacttgta tgtgtgtgtg tatatataat

7981 atgcatatat agttaccgtg ctaaaatggt taccagcagg ttttgagaga gaatgctgca

8041 tcagaaaagt gtcagttgcc acctcattct ccctgattta ggttcctgac actgattcct

8101 ttctctctcg tttttgaccc ccattgggtg tatcttgtct atgtacagat attttgtaat

8161 atattaaatt tttttctttc agtttataaa aatggaaagt ggagattgga aaattaaata

8221 tttcctgtta ctataccact tttgctccat tgcatt

By "Cyclin Dependent Kinase 12 (CDK12)" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. NP_057591, version NP 057591.2, incorporated herein by reference, as reproduced below (SEQ ID NO: 37):

1 mpnserhggk kdgsggasgt lqpssgggss nsrerhrlvs khkrhkskhs kdmglvtpea

61 aslgtvikpl veyddissds dtfsddmafk ldrrenderr gsdrsdrlhk hrhhqhrrsr

121 dllkakqtek eksqevssks gsmkdrisgs skrsneetdd ygkaqvakss skesrssklh

181 kektrkerel ksghkdrsks hrkretpksy ktvdspkrrs rsphrkwsds skqddspsga

241 sygqdydlsp srshtssnyd sykkspgsts rrqsvsppyk epsayqsstr spspysrrqr

301 svspysrrrs ssyersgsys grspspygrr rssspflskr slsrsplpsr ksmksrsrsp

361 aysrhssshs kkkrsssrsr hssispvrlp lnsslgaels rkkkeraaaa aaakmdgkes

421 kgspvflprk enssveakds gleskklprs vkleksapdt elvnvthlnt evknssdtgk

481 vkldensekh lvkdlkaqgt rdskpialke eivtpketet seketppplp tiaspppplp

541 tttpppqtpp lpplppipal pqqpplppsq pafsqvpass tstlppsths ktsavssqan

601 sqppvqvsvk tqvsvtaaip hlktstlppl plppllpgdd dmdspketlp skpvkkekeq

661 rtrhlltdlp lppelpggdl sppdspepka itppqqpykk rpkiccpryg errqtesdwg

721 krcvdkfdii giigegtygq vykakdkdtg elvalkkvrl dnekegfpit aireikilrq

781 lihrs vnmk eivtdkqdal dfkkdkgafy lvfeymdhdl mgllesglvh fsedhiks fm

841 kqlmegleyc hkknflhrdi kcsnillnns gqikladfgl arlynseesr pytnkvitlw

901 yrppelllge erytpaidvw scgcilgelf tkkpifqanl elaqlelisr lcgspcpavw

961 pdviklpyfn tmkpkkqyrr rlreefsfip saaldlldhm ltldpskrct aeqtlqsdf1

1021 kdvelskmap pdlphwqdch elwskkrrrq rqsg vveep ppsktsrket tsgtstepvk

1081 nsspappqpa pgkvesgagd aigladitqq lnqselavll nllqsqtdls ipqmaqllni

1141 hsnpemqqql ealnqsisal teatsqqqds etmapeeslk eapsapvilp saeqttleas

1201 stpadmqnil avllsqlmkt qepagsleen nsdknsgpqg prrtptmpqe eaaacpphil

1261 ppekrppepp gppppppppp lvegdlssap qelnpavtaa llqllsqpea eppghlpheh

1321 qalrpmeyst rprpnrtygn tdgpetgfsa idtdernsgp alteslvqtl vknrtfsgsl

1381 shlgesssyq gtgsvqfpgd qdlrfarvpl alhp vgqpf lkaegssnsv vhaetklqny

1441 gelgpgttga sssgaglhwg gptqssaygk lyrgptrvpp rggrgrgvpy

By "AKT3 nucleic acid molecule" is meant a polynucleotide encoding a AKT3 polypeptide. An exemplary AKT3 nucleic acid molecule is provided at NCBI Accession No. NM_005465, version NM_005465.4, incorporated herein by reference, and reproduced below (SEQ ID NO: 38):

1 gctgagtcat cactagagag tgggaagggc agcagcagca gagaatccaa accctaaagc 61 tgatatcaca aagtaccatt tctccaagtt gggggctcag aggggagtca tcatgagcga 121 tgttaccatt gtgaaagaag gttgggttca gaagagggga gaatatataa aaaactggag 181 gccaagatac ttccttttga agacagatgg ctcattcata ggatataaag agaaacctca 241 agatgtggat ttaccttatc ccctcaacaa cttttcagtg gcaaaatgcc agttaatgaa 301 aacagaacga ccaaagccaa acacatttat aatcagatgt ctccagtgga ctactgttat 361 agagagaaca tttcatgtag atactccaga ggaaagggaa gaatggacag aagctatcca 421 ggctgtagca gacagactgc agaggcaaga agaggagaga atgaattgta gtccaacttc 481 acaaattgat aatataggag aggaagagat ggatgcctct acaacccatc ataaaagaaa 541 gacaatgaat gattttgact atttgaaact actaggtaaa ggcacttttg ggaaagttat 601 tttggttcga gagaaggcaa gtggaaaata ctatgctatg aagattctga agaaagaagt 661 cattattgca aaggatgaag tggcacacac tctaactgaa agcagagtat taaagaacac 721 tagacatccc tttttaacat ccttgaaata ttccttccag acaaaagacc gtttgtgttt 781 tgtgatggaa tatgttaatg ggggcgagct gtttttccat ttgtcgagag agcgggtgtt 841 ctctgaggac cgcacacgtt tctatggtgc agaaattgtc tctgccttgg actatctaca 901 ttccggaaag attgtgtacc gtgatctcaa gttggagaat ctaatgctgg acaaagatgg 961 ccacataaaa attacagatt ttggactttg caaagaaggg atcacagatg cagccaccat 1021 gaagacattc tgtggcactc cagaatatct ggcaccagag gtgttagaag ataatgacta 1081 tggccgagca gtagactggt ggggcctagg ggttgtcatg tatgaaatga tgtgtgggag 1141 gttacctttc tacaaccagg accatgagaa actttttgaa ttaatattaa tggaagacat 1201 taaatttcct cgaacactct cttcagatgc aaaatcattg ctttcagggc tcttgataaa 1261 ggatccaaat aaacgccttg gtggaggacc agatgatgca aaagaaatta tgagacacag 1321 tttcttctct ggagtaaact ggcaagatgt atatgataaa aagcttgtac ctccttttaa 1381 acctcaagta acatctgaga cagatactag atattttgat gaagaattta cagctcagac 1441 tattacaata acaccacctg aaaaatatga tgaggatggt atggactgca tggacaatga 1501 gaggcggccg catttccctc aattttccta ctctgcaagt ggacgagaat aagtctcttt 1561 cattctgcta cttcactgtc atcttcaatt tattactgaa aatgattcct ggacatcacc 1621 agtcctagct cttacacata gcaggggcac cttccgacat cccagaccag ccaagggtcc 1681 tcacccctcg ccacctttca ccctcatgaa aacacacata cacgcaaata cactccagtt 1741 tttgtttttg catgaaattg tatctcagtc taaggtctca tgctgttgct gctactgtct 1801 tactattata gcaactttaa gaagtaattt tccaaccttt ggaagtcatg agcccaccat 1861 tgttcatttg tgcaccaatt atcatctttt gatcttttag tttttccctc agtgaaggct 1921 aaatgagata cactgattct aggtacattt tttaactttc tagaagagaa aaactaacta 1981 gactaagaag atttagttta taaattcaga acaagcaatt gtggaagggt ggtggcgtgc 2041 atatgtaaag cacatcagat ccgtgcgtga agtaggcata tatcactaag ctgtggctgg 2101 aattgattag gaagcatttg gtagaaggac tgaacaactg ttgggatata tatatatata 2161 tataattttt tttttttaaa ttcctggtgg atactgtaga agaagcccat atcacatgtg 2221 gatgtcgaga cttcacgggc aatcatgagc aagtgaacac tgttctacca agaactgaag 2281 gcatatgcac agtcaaggtc acttaaaggg tcttatgaaa caatttgagc cagagagcat 2341 ctttcccctg tgcttggaaa ccttttttcc ttcttgacat ttatcacctc tgatggctga 2401 agaatgtaga caggtataat gatactgctt ttcaccaaaa tttctacacc aaggtaaaca 2461 ggtgtttgcc ttatttaatt ttttactttc agttctacgt gaattagctt tttctcagat 2521 gttgaaactt tgaatgtcct tttatgattt tgtttatatt gcagtagtat ttatttttta 2581 gtgatgagaa ttgtatgtca tgttagcaaa cgcagctcca acttatataa aatagactta 2641 ctgcagttac ttttgaccca tgtgcaagga ttgtacacgc tgatgagaat catgcacttt 2701 ttctcctctg ttaaaaaaaa tgataaggct ctgaaatgga atatattggt tagaatttgg 2761 ctttgggaga agagatgctg ccatttaacc ccttggtact gaaaatgaga aaatccccaa 2821 ctatgcatgc caaggggtta atgaaacaaa tagctgttga cgtttgctca tttaagaatt 2881 tgaaacgtta tgatgacctg gcaacaaaaa gtaatgaaga aaattgagac ctgagtgaag 2941 ataagaaatg atctttacgt ggcaaaatga acacatcttg agtatttagg aaatgggcag 3001 tgaaggctaa gaacctggtg tgtttcttgg gatcatggta catttatcac tgaattaagc 3061 catcagggaa aaaacaacaa aaaaagagaa cacctccagc ttttcttttt ctgtatatac 3121 tcatgtcccc cagattccaa catttctcac tgaaaggggg catgtatgca aacctcatct 3181 ttctccttca ttaatgatga tcttcagatt aaaccctttg gtgctaggag ctgacaattt 3241 ccaaagcagc ctgtgaagtc ctaggggctg ggggccactc ttgcggcaag cagaaggcca 3301 tcctactccg cggagtgatc atggaaatgt attttagtta aactctgaca gctcccaaac 3361 ggaagactac agcatgacgt agtattatga ttgcattgta tgaaagagca agtgactttc 3421 taagtaggat gaatcatatt catatgcaga tgtcttagcc tcttgacgct ggaagtgtgg 3481 atttatagct atgaaaccac tgctggcagt gggtgggcca ctgggactga cgggggttaa 3541 agggcatttt actaaggcag ctaagacata ttcagacatc aacgttatcc ttctttttca 3601 tatttctacc tgagtgaagt tcatccttag tattgagtag gaagttacag taaatggtag 3661 ttcattctta cttacacaca tagctaatct tttttttttc acttggaatt atgttgaatg 3721 tttcattttg acaaaaaagt agactagaag gtatgttctt taagttgtct tgcatccatt 3781 atataagaaa gaaacaggtg agaggaagag cagaaagctg agactggctg atgttcagag 3841 cacttactcc tctagaggga aagcatgaca ccgaacacta agcacacagc tttttgttgt 3901 tttggttttt tctcccgcaa atcttaaagt gattcccatg accttggcca aggacacttc 3961 ttaaagatta atgactggca ctgacattgc cccaggcggg ccactcctca cactggctct 4021 cagttcccag ccatgcctgg ggctcagtca cttctattcc accctctgag actccattgg 4081 tgtcacacaa ggtgtcttct tggctttgat tttgagaatc ccctattttc acttccagat 4141 ctgtcagctg ccatggagga ataatagaaa accagaaatg cgtgtagagg gagatttcta 4201 aaacttccct tgtgtcgccc atagttgtag ttttgggttc tggcaggtgg aacaccctga 4261 aacctggaat cattctatga gaatacagtt cagactttgc agactccagc ccatactaac 4321 tgtcatgaag cttgacttct tgtcataatg cagccatctt ggaggaaatt ggccatttct 4381 gcttagatgg ttggcagggt cgcgctcagc tttgctttct acactaatta catagcatta 4441 ttcaagtatt gttttccatt tcccatccct gatttccagc ttcttaaagc tgactgttct 4501 tgcaggggcc acttgcttct cctagagtac aaaagtaagg gccttcctta ctaactgcag 4561 ggtctctcta ttacacctca acatacacac tttgctgcta ctgtttgtac tgtctacagt 4621 agaatttcct tatcttgctc ctggtagtgc attacaggca agcatgaaat gtaaagtatt 4681 tatttaaata aaaagaaaac ctctaaattg gtaattgaat tacctccctg tagctttata 4741 gtttgtgaca tttcttgacc ttgctagttc tttcattaga tctgcgcaag atctagtcat 4801 ctggttaagg attttaagca gatgcaacta taaacccaag aaactgtatt actattactg 4861 ttggtcatac taaacctgtc tatttcctga agtatatgac ccacaaggat gtggaataac 4921 taggagaaac tgtttttgta cactgtacat ccttagtatt tttacacgta tatgataggg 4981 atgaacatga ttttccttcg tacagacagc ttaaataaag cactatgtca atctgctact 5041 tctctgttta ttgttgttgg atgtggttct ataatccccc caaattaaat cttctttaat 5101 gaaaacatga tttttaatag ccccagctgg tattaaccta ccttgtataa aatgtgacag 5161 gaaaatatag aaataattcc ttgtagctca cacacacaca cataggggat catttttact 5221 tcagtgaaat ggcagtagtg cggttgtgca aactttgatg aacggctgct tctgagggga 5281 aacgctgacc tctcagcact ggatttagga tggatgtact gtgaagccag ggatgaagga 5341 ggtctcagac cctggggaca ttcagacccg aatcatctat acaacacacg gtttggaccc 5401 agaatctgaa ggaatgtagc ttttcattaa cgtcttcctg ataatgtact gctctgcata 5461 tttcctttct tagagtgtat ttctaacaac atgtcatggc aaattaacaa acttagacgt 5521 gggtgatgta gatgggtagg atggctggac tgcagtctga cttcacgttg aatcattctg 5581 gatggggcct ttttctgatt ttacctcata aagctactat tgtagaaact tggctttgct 5641 cctgtgacga agccagacag aggaatggct tttgggacca gagtgagtca agcatgtatg 5701 tgtatgtcac acggccaaat ttgagggcat tctcacatgt gctcttctct caaaaccact 5761 ggggttgaca gatccaggag gctaaaaaaa agtgacctct ataattcttt aaaggtgcta 5821 tttttagaat attgtataat ttattcacag tatatctaaa acagaattaa ggacaattaa 5881 aatatcttat gtgacagcct ttatgtctag cacatttgat gaaataaaaa acttctgaat 5941 ctgaatagaa gttctactgt ttcaggcttg aaccttttac atgctcaaga gattcaaatg 6001 gtctctgtgt gtagatcatg ccaccgcctc caaagcctaa tccacatcac ttctgagagg 6061 caaggctgag catatggtga catcagctct gtgttgagat ggtgatgagg atgatggctc 6121 gctggccagg cagggcagcc gaaggtcagg gacctgtcct aactaactgc agccttgcct 6181 ttagtgtttg tcattctcag atacaacacg gtatgtccag tgtccgtttt tattacttta 6241 aagcatttga gggcttaatt gtgtatagta gaaatactat tttagacaaa taattatctg 6301 tgtacagata tttgatatac tctaagtaaa ttttctaatt tcactaagta cgtttttagg 6361 ctcctctcaa atactgcgta ttgaagaaaa aaatctgaca ccaccgagcc aaagatgctt 6421 ttttgtctgt tttcgttgtt taacagaatg gaaagagtaa tgcatagtgc ttcctggtgt 6481 ctcctgattg attgattgtg cacaaagtag gacgataaat aaataaaatg gagtctgatg 6541 ggacattgat taaaggtgaa ggatgattga tatatagatc atgaaaagaa aaatgaatgg 6601 caggaaaaaa agtttggtcc ttaatatact ttggcctagt taaaatatgt gcctttttgg 6661 tgtgttttgt tcatcactac aagataaaaa ggaaacatta caactcaagt ctttaaaaag 6721 ttcatttatt gaaaatcata tgtataacct agcatacgaa tgagcagatt taaacacata 6781 acttcaagcc atttctgaaa acatacacca ggagctctgc tcagctagag tcagactcca 6841 gctccagccc gactgcgtgc ggggacagcg cccgcgttga tgaggaccag ccccactgca 6901 ggctgaggcg gtgtcaccct gggaaggtcg tggtgcgttg tggcatatta agtctaaacc

6961 agatgaatgt aaatatctct ttgtaaatca tttatttcac tctgttccat ccaggtcagc

7021 aatcagattg tggcatgctg ggtaactgga aaaaataata aaaagtaagt ttcaatagct

7081 caaaaaaaaa a

By "AKT3" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. CAB53537, version CAB53537.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 39):

1 msdvtivkeg wvqkrgeyik nwrpryfllk tdgsfigyke kpqdvdlpyp lnnfsvakcq 61 lmkterpkpn tfiirclqwt tviertfhvd tpeereewte aiqavadrlq rqeeermncs 121 ptsqidnige eemdastthh krktmndfdy lkllgkgtfg kvilvrekas gkyyamkilk 181 keviiakdev ahtltesrvl kntrhpflts lkysfqtkdr lcfvmeyvng gelffhlsre 241 rvfsedrtrf ygaeivsald ylhsgkivyr dlklenlmld kdghikitdf glckegitda 301 atmktfcgtp eylapevled ndygravdww glgvvmyemm cgrlpfynqd heklfelilm 361 edikfprtls sdaksllsgl likdpnkrlg ggpddakeim rhsffsgvnw qdvydkklvp 421 pfkpqvtset dtryfdeeft aqtititppe kydedgmdcm dnerrphfpq fsysasgre

By "Tyrosine-Protein Kinase Receptor 3 (TYR03) nucleic acid molecule" is meant a polynucleotide encoding a TYR03 polypeptide. An exemplary TYR03 nucleic acid molecule is provided at NCBI Accession No. X72886, version X72886.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 40):

1 accctgggcc ggatgttggg caaaggagag tttggttcag tgcgggaggc ccagctgaag 61 caagaggatg gctcctttgt gaaagtggct gtgaagatgc tgaaagctga catcattgcc 121 tcaagcgaca ttgaagagtt cctcagggaa gcagcttgca tgaaggagtt tgaccatcca 181 cacgtggcca aacttgttgg ggtaagcctc cggagcaggg ctaaaggccg tctccccatc 241 cccatggtca tcttgccctt catgaagcat ggggacctgc atgccttcct gctcgcctcc 301 cggattgggg agaacccctt taacctaccc ctccagaccc tgatccggtt catggtggac 361 attgcctgcg gcatggagta cctgagctct cggaacttca tccaccgaga cctggctgct 421 cggaattgca tgctggcaga ggacatgaca gtgtgtgtgg ctgacttcgg actctcccgg 481 aagatctaca gtggggacta ctatcgtcaa ggctgtgcct ccaaactgcc tgtcaagtgg 541 ctggccctgg agagcctggc cgacaacctg tatactgtgc agagtgacgt gtgggcgttc 601 ggggtgacca tgtgggagat catgacacgt gggcagacgc catatgctgg catcgaaaac 661 gctgagattt acaactacct cattggcggg aaccgcctga aacagcctcc ggagtgtatg 721 gaggacgtgt atgatctcat gtaccagtgc tggagtgctg accccaagca gcgcccgagc 781 tttacttgtc tgcgaatgga actggagaac atcttg

By "Tyrosine-Protein Kinase Receptor 3 (TYR03)" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAH51756, version AAH51756.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 41):

1 malrrsmgrp glpplplppp prlglllaal aslllpesaa aglklmgapv kltvsqgqpv 61 klncsvegme epdiqwvkdg a vqnldqly ipvseqhwig flslksvers dagrywcqve 121 dggeteisqp vwltvegvpf ftvepkdlav ppnapfqlsc eavgppepvt ivwwrgttki 181 ggpapspsvl nvtgvtqstm fsceahnlkg lassrtatvh lqalpaapfn itvtklsssn 241 asvawmpgad grallqsctv qvtqapggwe vlavvvpvpp ftcllrdlvp atnyslrvrc 301 analgpspya dwvpfqtkgl apasapqnlh airtdsglil eweevipeap legplgpykl 361 swvqdngtqd eltvegtran ltgwdpqkdl ivrvcvsnav gcgpwsqplv vsshdragqq 421 gpphsrtswv pvvlgvltal vtaaalalil lrkrrketrf gqafdsvmar gepavhfraa 481 rsfnrerper ieatldslgi sdelkekled vlipeqqftl grmlgkgefg svreaqlkqe 541 dgsfvkvavk mlkadiiass dieeflreaa cmkefdhphv aklvgvslrs rakgrlpipm 601 vilpfmkhgd lhafllasri genpfnlplq tlirfmvdia cgmeylssrn fihrdlaarn 661 cmlaedmtvc vadfglsrki ysgdyyrqgc asklpvkwla lesladnlyt vqsdvwafgv 721 tmweimtrgq tpyagienae iynyliggnr lkqppecmed vydlmyqcws adpkqrpsft 781 clrmelenil gqlsvlsasq dplyiniera eeptaggsle lpgrdqpysg agdgsgmgav 841 ggtpsdcryi ltpgglaeqp gqaehqpesp lnetqrllll qqgllphssc

By "Ephrin Type-A Receptor 5 (EPHA5) nucleic acid molecule" is meant a

polynucleotide encoding a EPHA5 polypeptide. An exemplary EPHA5 nucleic acid molecule is provided at NCBI Accession No. NM_004439, version NM_004439.7, incorporated herein by reference, and reproduced below (SEQ ID NO: 42):

1 ccagacacag caggagcgcg ccttggcggt gcagtttcaa cctcgacttc gcagccgcgc

61 acacaccgcc tgctccccga gcagcgggaa ccgcagcagc tcctgggccg ccgcagcgca 121 gctccgcgct cctaccggcc ggcagccgtc agtccctccc ctcttcagca ctcagcccgc 181 agctatttcc ttctgccagt ctctttgaac tctggatctt tgcttttgct cgctgctctc 241 ctgtttttca ttctccacat tttctcaagt cctctttctt tatccttagc caccctgctt 301 ttttcctcct tttttaaaaa atcggagatt tcgtcttaaa atgatttgcc ttccttacct 361 tcgtccattt caacactgaa ggctgcaaag aaccttcacc tttcccctag tggtatttaa 421 aaattctcaa tccgtaaaaa gtctttttga aaggcaaagg aacaggaccc aggaccctct 481 cgacaccctt gatccgagtc cagatctgca ctagcaacca gaactaatat ttcatttaac 541 cccaccaaag ggggaggcga gaggagccag aagcaaactt cagctgtctc agccggatcc 601 gtggttccta catttggagg agccgcgtgc cagaaggcgt aggaccccaa ggggggacaa 661 ggaggactcc cgagtctccc ttcgccgctc tgcgaggccg aagcggtgga ctgagccgct 721 cgggacagcg gcaccggagg aggctcggag aagatgcggg gctcggggcc ccggggtgcg 781 ggacgccggc ggcccccaag cggcggcggc gacaccccca tcaccccagc gtccctggcc 841 ggctgctact ctgcacctcg acgggctccc ctctggacgt gccttctcct gtgcgccgca 901 ctccggaccc tcctggccag ccccagcaac gaagtgaatt tattggattc acgcactgtc 961 atgggggacc tgggatggat tgcttttcca aaaaatgggt gggaagagat tggtgaagtg 1021 gatgaaaatt atgcccctat ccacacatac caagtatgca aagtgatgga acagaatcag 1081 aataactggc ttttgaccag ttggatctcc aatgaaggtg cttccagaat cttcatagaa 1141 ctcaaattta ccctgcggga ctgcaacagc cttcctggag gactggggac ctgtaaggaa 1201 acctttaata tgtattactt tgagtcagat gatcagaatg ggagaaacat caaggaaaac 1261 caatacatca aaattgatac cattgctgcc gatgaaagct ttacagaact tgatcttggt 1321 gaccgtgtta tgaaactgaa tacagaggtc agagatgtag gacctctaag caaaaaggga 1381 ttttatcttg cttttcaaga tgttggtgct tgcattgctc tggtttctgt gcgtgtatac 1441 tataaaaaat gcccttctgt ggtacgacac ttggctgtct tccctgacac catcactgga 1501 gctgattctt cccaattgct cgaagtgtca ggctcctgtg tcaaccattc tgtgaccgat 1561 gaacctccca aaatgcactg cagcgccgaa ggggagtggc tggtgcccat cgggaaatgc 1621 atgtgcaagg caggatatga agagaaaaat ggcacctgtc aagtgtgcag acctgggttc 1681 ttcaaagcct cacctcacat ccagagctgc ggcaaatgtc cacctcacag ttatacccat 1741 gaggaagctt caacctcttg tgtctgtgaa aaggattatt tcaggagaga gtctgatcca 1801 cccacaatgg catgcacaag acccccctct gctcctcgga atgccatctc aaatgttaat 1861 gaaactagtg tctttctgga atggattccg cctgctgaca ctggtggaag gaaagacgtg 1921 tcatattata ttgcatgcaa gaagtgcaac tcccatgcag gtgtgtgtga ggagtgtggc 1981 ggtcatgtca ggtaccttcc ccggcaaagc ggcctgaaaa acacctctgt catgatggtg 2041 gatctactcg ctcacacaaa ctataccttt gagattgagg cagtgaatgg agtgtccgac 2101 ttgagcccag gagcccggca gtatgtgtct gtaaatgtaa ccacaaatca agcagctcca 2161 tctccagtca ccaatgtgaa aaaagggaaa attgcaaaaa acagcatctc tttgtcttgg 2221 caagaaccag atcgtcccaa tggaatcatc ctagagtatg aaatcaagta ttttgaaaag 2281 gaccaagaga ccagctacac gattatcaaa tctaaagaga caactattac tgcagagggc 2341 ttgaaaccag cttcagttta tgtcttccaa attcgagcac gtacagcagc aggctatggt 2401 gtcttcagtc gaagatttga gtttgaaacc accccagtgt ttgcagcatc cagcgatcaa 2461 agccagattc ctgtaattgc tgtgtctgtg acagtgggag tcattttgtt ggcagtggtt 2521 atcggcgtcc tcctcagtgg aagttgctgc gaatgtggct gtgggagggc ttcttccctg 2581 tgcgctgttg cccatccaag cctaatatgg cggtgtggct acagcaaagc aaaacaagat 2641 ccagaagagg aaaagatgca ttttcataat gggcacatta aactgccagg agtaagaact 2701 tacattgatc cacataccta tgaggatccc aatcaagctg tccacgaatt tgctaaggag 2761 atagaagcat catgtatcac cattgagaga gttattggag caggtgaatt tggtgaagtt 2821 tgtagtggac gtttgaaact accaggaaaa agagaattac ctgtggctat caaaaccctt 2881 aaagtaggct atactgaaaa gcaacgcaga gatttcctag gtgaagcaag tatcatggga 2941 cagtttgatc atcctaacat catccattta gaaggtgtgg tgaccaaaag taaaccagtg 3001 atgatcgtga cagagtatat ggagaatggc tctttagata catttttgaa gaaaaacgat 3061 gggcagttca ctgtgattca gcttgttggc atgctgagag gtatctctgc aggaatgaag 3121 tacctttctg acatgggcta tgtgcataga gatcttgctg ccagaaacat cttaatcaac 3181 agtaaccttg tgtgcaaagt gtctgacttt ggactttccc gggtactgga agatgatccc 3241 gaggcagcct acaccacaag gggaggaaaa attccaatca gatggactgc cccagaagca 3301 atagctttcc gaaagtttac ttctgccagt gatgtctgga gttatggaat agtaatgtgg 3361 gaagttgtgt cttatggaga gagaccctac tgggagatga ccaatcaaga tgtgattaaa 3421 gcggtagagg aaggctatcg tctgccaagc cccatggatt gtcctgctgc tctctatcag 3481 ttaatgctgg attgctggca gaaagagcga aatagcaggc ccaagtttga tgaaatagtc 3541 aacatgttgg acaagctgat acgtaaccca agtagtctga agacgctggt taatgcatcc 3601 tgcagagtat ctaatttatt ggcagaacat agcccactag gatctggggc ctacagatca 3661 gtaggtgaat ggctagaggc aatcaagatg ggccggtata cagagatttt catggaaaat 3721 ggatacagtt caatggacgc tgtggctcag gtgaccttgg aggatttgag acggcttgga 3781 gtgactcttg tcggtcacca gaagaagatc atgaacagcc ttcaagaaat gaaggtgcag 3841 ctggtaaacg gaatggtgcc attgtaactt catgtaaatg tcgcttcttc aagtgaatga 3901 ttctgcactt tgtaaacagc actgagattt attttaacaa aaaaaggggg aaaagggaaa 3961 acagtgattt ctaaacctta gaaaacattt gcctcagcca cagaatttgt aatcatggtt 4021 ttactgaagt atccagttct tagtccttag tcttcatttt tcatgaagca aacatatctt 4081 gcattaaaag ggacatgaag ttagacatca tcttaagtta caacaacaga atccttccca 4141 ctacttctac aaaattttgt acatgaaata tataattata tagcactttt atagactgaa 4201 ttaaggcaac ccctttcaaa acttccaggg atctacttga aaggaaatgt tttatagcca 4261 tttgtgagct aacaaaagct acagtttact gaagtttact tcaagtctta attgtctaca 4321 aaagtgtatt gaagagcaat atgattagat tatttcttaa tagatatctt cttttgtaat 4381 tttaaaatgc tgttacacag cgttaagtta tagaaactag tgtgtaaaca tgttgcttga 4441 tcaagaaaaa gtacaataca gggtgtatat ttattttttg tgttataaag tttactttta 4501 gttgctcttc tagagattat taggtaataa atgtgtatat actgtataat ttgcaatata 4561 cccaggaact gatttaagat ggaattgtgt gtgtgtttgc ttgcacatgt gtgtgttacc 4621 attctgcttg catttctaat agtgtttcaa ttttagcaac atataggagc aagtgttcca 4681 gaatgtaata tgaataggag aaatagggaa gcagtaaaac aaaatttaac acaagcttgt 4741 gtctcttttc ctctcatgtg tccaaaagct aatctcttta ttcactaaaa ggaaatgtgt 4801 ataagactaa atcccctttg gctttttaaa aacattttgt gatatcagtg acaatgcagt 4861 tcttctagcc attaatcttg tacccctgta agaaatttca cctttctgag tcctgaaaag 4921 tatcttgtca agcaaagttg acaccgaagg gcacattttc agcaggatgt agaaggattt 4981 aactgtgcag gcttctgtta atgttgttaa atccaggcac atagcacgaa gcatcaccct 5041 taagtgttaa tccgttgtaa ccattcccat tttgactcag ttctagaaat tttgactcta 5101 aggcagcaat ggaattatga caaatatata tatatacaca tacacacaca caaatatata 5161 tatatgctta tttcccttca gaattttatt tcaataattg ataagtttta tttttaatgg 5221 atgtttattc atttgttaat ttcagtttgt attcagttac atggctttga gtttatttta 5281 ttgaaccaat ccaaggtatt atgtaattag gcttattaag gaatataaca tatcttctat 5341 gtatgctcta tataccacac atgatgaaga cttatatttg tgatacaaat tatgcagtgt 5401 attagaaatt gtccaaaact gagctcctga gataaccaac atttttgtat tattacactt 5461 tataaaaatt atggaatgtc atgtgattag tcaaaatcag agcatttaaa taaaatacag 5521 cctactcatg attatcaatg ttaatagtac agtagatcag agaagcctgg attaatgaaa 5581 tttatcagaa atatagccac gttatattaa tggtattctt ttttccaagc acttgtttta 5641 tttgaatatt agcacatata agaataaact tttgggaaat aactacttcc tattctgccc 5701 accctttact taagcagtaa agtacctctt ttctaaagaa ccaggaaaca tcaaaataac 5761 cacaacaaaa atctaaacag ttttcaaata atacattcag ttctcataat aataagggct 5821 atataccatg ggtccacaca aactgtgctg caaacatata atctattagt ttagtaaaat 5881 ttccagagat gtgaatttaa tattttcttg ataaaatcat aaaaaagcat caccattatt 5941 aaagatgcat ttgttcattt caataaacag aaattaatga aacaaattac ttttgtacaa 6001 aataaatgat gattatgtgg cactttattg aattttatag aaatattcca atctaagtga 6061 taaagtaaca actgagaaac ttcagaatga aagccttatt gattcaaaag gaaaattacc 6121 atatgcttta gggctgatga acttgccaca attgcttaga cataacaaag atattttcta 6181 cattgttttc caactttata tgctgaaaga taattaggag gtccgggtgg agtgtatcta 6241 aatgactaga actttaagtt gcaatataga ttttctcttt ttaacaaagg aagaatataa 6301 attaatttgg atcaaattta tttgccttct ttgcaatctt ggtgatcatt ttggaaagta 6361 aattgaaagg aaagttaaat agccacatag ttttcttttg catctcaatt tggttgagaa 6421 tttctaagga aggttaatat gactttagaa tgaataaaga gactgtcaaa caacaagtac 6481 tctccctaaa aaagaggaaa gaatgccttc ctaaaatatt tgtttcctat tgtatatgtc 6541 accaattgga aaatgccaat ctcatctgca agtgtgagaa atggaacaag gagaggagcc 6601 aactctagta aaatgtagtg aggccataat ccaaggcaaa gtttaacaaa tcttgattgt 6661 gtaatttctt attctgttga ttcattttgt cacccaccag actaccaaaa ataaagcaca 6721 gacatgacaa ttttagtata tacaacagat ttggcaaata atacataatc ttattgtaat 6781 tgccagtagc aatatcttaa ggggcatgag tctttcacag tattggactt tgaaaaattg 6841 gtacagggag taattttcaa cagactgata cttgaagtca ctcaattact actttgacct 6901 atcataattt gtttctaatc ttgtcatttg ctcatatata ctattaatat aaccagttgc 6961 tcttttcata atgtactatt tctatgtaaa gatgtaaaga ttttgatttt cctatgtttt 7021 aaaattgtaa tgtgcatgat cttttatttc ttgtgtttca attaccaagt cagtattcta 7081 aggttgtaag attactgtaa aaattatatt ccaatatttt atctaaactt aagtgtggcc 7141 agcattattg gtttcagagt tgcctaaaca acacagaata tccttaattc agtttgtaaa 7201 agagttttga ccagtgtaaa aaagtgaaga cacagtttca tgtttcaagt ttaaaatgga 7261 aagttaatat catttgagca cttatgtgtt ttcatgtctt ctacttaatc tgtatgtgaa 7321 agtaaaatat ttttaacacc attatttatg ccatgtaaag tgggttctca gaagcacaag 7381 cacagatatt taccttctga agacattttt ggctataaaa gtgcattgga tggcaaacac 7441 tatttgagtc aggtgttaga aatttattag atgtattata ttacatagta gaataaggtc 7501 cctttctcat tttgtttttc ctaaaaataa gaaaaaaaag aagtgtgata gtaaactgtc 7561 tttgctaatg tcctgccaga tgtttaactt caaattaagc aaaaagtagg tacaatatga 7621 tgatgatcct gatgatgatg atgatgatga tgatgaagaa taaatggaat caaaatgcta 7681 gctttatttg acattaataa gtaaaataag tcatcatttt ttcaactctg tagcacagct 7741 gtttacattg aataattttc tctattgtgc tgttaattat atagtaatgt atcaaaatag 7801 aaataggaac tttcttttca tttcttattc atttatgaag aaatcttttc acacataaat 7861 gattgaatgt tcttgttcag aatgaccaca taatcattgc ttagaagaag ataccaattc 7921 ttttaaaaga aaaaaagtct gtttataata atcagaatca aatgttgttt gtttcttcta 7981 aacgttaatg gagaaaattg aaggtggtaa aatgtcatgt ttattcaggc tgggaactgt 8041 attcacagta gaagtttcag tggtcaacat atctatgact ctttaggctg ctgtagtttt 8101 acagtcaatt atttaaaagt gagtagttac atttataaga gcctgagaat acttagactc 8161 agtcatttgt tagtattttt accaaaatct cttagtttca gacatgtcag aagcagctat 8221 atagcatatc ttattctatg atatacatca ggctatctca agttcctgtc tcacagttaa 8281 ttcaaagaag gattaggatt tctgtatttt ttctcatttg aatctttatg tgcatttggt 8341 ttgtgtacat gctttttgta gtgtaagata tgaaatttta tattttttca gaaaataaaa 8401 accctttgaa tacagttaaa aaaaaaaaaa aaaaa By "Ephrin Type-A Receptor 5 (EPHA5)" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAI43428, version AAI43428.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 43):

1 mrgsgprgag rrrppsgggd tpitpaslag cysaprrapl wtclllcaal rtllaspsne 61 vnlldsrtvm gdlgwiafpk ngweeigevd enyapihtyq vckvmeqnqn nwlltswisn 121 egasrifiel kftlrdcnsl pgglgtcket fnmyyfesdd qngrnikenq yikidtiaad 181 esfteldlgd rvmklntevr dvgplskkgf ylafqdvgac ialvsvrvyy kkcps vrhl 241 avfpdtitga dssqllevsg scvnhsvtde ppkmhcsaeg ewlvpigkcm ckagyeekng 301 tcqvcrpgff kasphiqscg kcpphsythe eastscvcek dyfrresdpp tmactrppsa 361 prnaisnvne tsvflewipp adtggrkdvs yyiackkcns hagvceecgg hvrylprqsg 421 lkntsvmmvd llahtnytfe ieavngvsdl spgarqyvsv nvttnqaaps pvtnvkkgki 481 aknsislswq epdrpngiil eyeikyfekd qetsytiiks kettitaegl kpasvyvfqi 541 rartaagygv fsrrfefett pvsvaassdq sqipviavsv tvgvilla v igvllsgrrc 601 gyskakqdpe eekmhfhngh iklpgvrtyi dphtyedpnq avhefakeie ascitiervi 661 gagefgevcs grlklpgkre lpvaiktlkv gytekqrrdf lgeasimgqf dhpniihleg 721 vtkskpvmi vteymengsl dtflkkndgq ftviqlvgml rgisagmkyl sdmgyvhrdl 781 aarnilinsn lvckvsdfgl srvleddpea ayttrggkip irwtapeaia frkftsasdv 841 wsygivmwev vsygerpywe mtnqdvikav eegyrlpspm dcpaalyqlm ldcwqkerns 901 rpkfdeivnm ldklirnpss lktlvnascr vsnllaehsp lgsgayrsvg ewleaikmgr 961 yteifmengy ssmdavaqvt ledlrrlgvt lvghqkkimn slqemkvqlv ngmvpl

By "Neurotrophic Receptor Tyrosine Kinase 3 (NTRK3) nucleic acid molecule" is meant a polynucleotide encoding a NTRK3 polypeptide. An exemplary NTRK3 nucleic acid molecule is provided at NCBI Accession No. NM_001012338, version NM_001012338.2, incorporated herein by reference, and reproduced below (SEQ ID NO: 44):

1 acatttctgc agccgcgcgg cgagccattc gcggcggctg ctgcagctcc tactgcatct

61 tccttctctt cctttcctcg ggctccggtc tcggagtcgg agagcgcgcc tcgcttccag 121 agcccccgga cccggcgagt cagcgatcgc cgagccggcc accatgcccg gcagaccgcg 181 ccactaggcg ctcctcgcgg ctcccacccg gcggcggcgg cggcggcggc ggcgtccgcg 241 atggtttcag acgctgaagg attttgcatc tgatcgctcg gcgtttcaaa gaagcagcga 301 tcggagatgg atgtctctct ttgcccagcc aagtgtagtt tctggcggat tttcttgctg 361 ggaagcgtct ggctggacta tgtgggctcc gtgctggctt gccctgcaaa ttgtgtctgc 421 agcaagactg agatcaattg ccggcggccg gacgatggga acctcttccc cctcctggaa 481 gggcaggatt cagggaacag caatgggaac gccagtatca acatcacgga catctcaagg 541 aatatcactt ccatacacat agagaactgg cgcagtcttc acacgctcaa cgccgtggac 601 atggagctct acaccggact tcaaaagctg accatcaaga actcaggact tcggagcatt 661 cagcccagag cctttgccaa gaacccccat ttgcgttata taaacctgtc aagtaaccgg 721 ctcaccacac tctcgtggca gctcttccag acgctgagtc ttcgggaatt gcagttggag 781 cagaactttt tcaactgcag ctgtgacatc cgctggatgc agctctggca ggagcagggg 841 gaggccaagc tcaacagcca gaacctctac tgcatcaacg ctgatggctc ccagcttcct 901 ctcttccgca tgaacatcag tcagtgtgac cttcctgaga tcagcgtgag ccacgtcaac 961 ctgaccgtac gagagggtga caatgctgtt atcacttgca atggctctgg atcacccctt 1021 cctgatgtgg actggatagt cactgggctg cagtccatca acactcacca gaccaatctg 1081 aactggacca atgttcatgc catcaacttg acgctggtga atgtgacgag tgaggacaat 1141 ggcttcaccc tgacgtgcat tgcagagaac gtggtgggca tgagcaatgc cagtgttgcc 1201 ctcactgtct actatccccc acgtgtggtg agcctggagg agcctgagct gcgcctggag 1261 cactgcatcg agtttgtggt gcgtggcaac cccccaccaa cgctgcactg gctgcacaat 1321 gggcagcctc tgcgggagtc caagatcatc catgtggaat actaccaaga gggagagatt 1381 tccgagggct gcctgctctt caacaagccc acccactaca acaatggcaa ctataccctc 1441 attgccaaaa acccactggg cacagccaac cagaccatca atggccactt cctcaaggag 1501 ccctttccag agagcacgga taactttatc ttgtttgacg aagtgagtcc cacacctcct 1561 atcactgtga cccacaaacc agaagaagac acttttgggg tatccatagc agttggactt 1621 gctgcttttg cctgtgtcct gttggtggtt ctcttcgtca tgatcaacaa atatggtcga 1681 cggtccaaat ttggaatgaa gggtcccgtg gctgtcatca gtggtgagga ggactcagcc 1741 agcccactgc accacatcaa ccacggcatc accacgccct cgtcactgga tgccgggccc 1801 gacactgtgg tcattggcat gactcgcatc cctgtcattg agaaccccca gtacttccgt 1861 cagggacaca actgccacaa gccggacacg tatgtgcagc acattaagag gagagacatc 1921 gtgctgaagc gagaactggg tgagggagcc tttggaaagg tcttcctggc cgagtgctac 1981 aacctcagcc cgaccaagga caagatgctt gtggctgtga aggccctgaa ggatcccacc 2041 ctggctgccc ggaaggattt ccagagggag gccgagctgc tcaccaacct gcagcatgag 2101 cacattgtca agttctatgg agtgtgcggc gatggggacc ccctcatcat ggtctttgaa 2161 tacatgaagc atggagacct gaataagttc ctcagggccc atgggccaga tgcaatgatc 2221 cttgtggatg gacagccacg ccaggccaag ggtgagctgg ggctctccca aatgctccac 2281 attgccagtc agatcgcctc gggtatggtg tacctggcct cccagcactt tgtgcaccga 2341 gacctggcca ccaggaactg cctggttgga gcgaatctgc tagtgaagat tggggacttc 2401 ggcatgtcca gagatgtcta cagcacggat tattacaggc tctttaatcc atctggaaat 2461 gatttttgta tatggtgtga ggtgggagga cacaccatgc tccccattcg ctggatgcct 2521 cctgaaagca tcatgtaccg gaagttcact acagagagtg atgtatggag cttcggggtg 2581 atcctctggg agatcttcac ctatggaaag cagccatggt tccaactctc aaacacggag 2641 gtcattgagt gcattaccca aggtcgtgtt ttggagcggc cccgagtctg ccccaaagag 2701 gtgtacgatg tcatgctggg gtgctggcag agggaaccac agcagcggtt gaacatcaag 2761 gagatctaca aaatcctcca tgctttgggg aaggccaccc caatctacct ggacattctt 2821 ggctagtggt ggctggtggt catgaattca tactctgttg cctcctctct ccctgcctca 2881 catctccctt ccacctcaca actccttcca tccttgactg aagcgaacat cttcatataa 2941 actcaagtgc ctgctacaca tacaacactg aaaaaaggaa aaaaaaagaa agaaaaaaaa 3001 accc

By "Neurotrophic Receptor Tyrosine Kinase 3 (NTRK3)" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No.

AAH13693, version AAH13693.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 45):

1 mdvslcpakc sfwrifllgs v ldyvgsvl acpancvcsk teincrrpdd gnlfpllegq

61 dsgnsngnas initdisrni tsihienwrs lhtlnavdme lytglqklti knsglrsiqp

121 rafaknphlr yinlssnrlt tlswqlfqtl slrelqleqn ffncscdirw mqlwqeqgea

181 klnsqnlyci nadgsqlplf rmnisqcdlp eisvshvnlt vregdnavit cngsgsplpd

241 vdwivtglqs inthqtnlnw tnvhainltl vnvtsedngf tltciaenvv gmsnasvalt

301 vyyppr vsl eepelrlehc ief vrgnpp ptlhwlhngq plreskiihv eyyqegeise

361 gcllfnkpth ynngnytlia knplgtanqt inghflkepf pestdnfilf devsptppit

421 vthkpeedtf gvsiavglaa facvll vlf vminkygrrs kfgmkgpvav isgeedsasp

481 lhhinhgitt pssldagpdt vigmtripv ienpqyfrqg hnchkpdtwv fsnidnhgil

541 nlkdnrdhlv psthyiyeep evqsgevsyp rshgfreiml npislpghsk plnhgiyved

601 vnvyfskgrh gf By "Androgen Receptor (AR) nucleic acid molecule" is meant a polynucleotide encoding a AR polypeptide. An exemplary AR nucleic acid molecule is provided at NCBI Accession No. NM_000044, version NM_000044.4, incorporated herein by reference, and reproduced below (SEQ ID NO: 46):

1 gcggagagaa ccctctgttt tcccccactc tctctccacc tcctcctgcc ttccccaccc

61 cgagtgcgga gccagagatc aaaagatgaa aaggcagtca ggtcttcagt agccaaaaaa 121 caaaacaaac aaaaacaaaa aagccgaaat aaaagaaaaa gataataact cagttcttat 181 ttgcacctac ttcagtggac actgaatttg gaaggtggag gattttgttt ttttctttta 241 agatctgggc atcttttgaa tctacccttc aagtattaag agacagactg tgagcctagc 301 agggcagatc ttgtccaccg tgtgtcttct tctgcacgag actttgaggc tgtcagagcg 361 ctttttgcgt ggttgctccc gcaagtttcc ttctctggag cttcccgcag gtgggcagct 421 agctgcagcg actaccgcat catcacagcc tgttgaactc ttctgagcaa gagaagggga 481 ggcggggtaa gggaagtagg tggaagattc agccaagctc aaggatggaa gtgcagttag 541 ggctgggaag ggtctaccct cggccgccgt ccaagaccta ccgaggagct ttccagaatc 601 tgttccagag cgtgcgcgaa gtgatccaga acccgggccc caggcaccca gaggccgcga 661 gcgcagcacc tcccggcgcc agtttgctgc tgctgcagca gcagcagcag cagcagcagc 721 agcagcagca gcagcagcag cagcagcagc agcagcagca gcaagagact agccccaggc 781 agcagcagca gcagcagggt gaggatggtt ctccccaagc ccatcgtaga ggccccacag 841 gctacctggt cctggatgag gaacagcaac cttcacagcc gcagtcggcc ctggagtgcc 901 accccgagag aggttgcgtc ccagagcctg gagccgccgt ggccgccagc aaggggctgc 961 cgcagcagct gccagcacct ccggacgagg atgactcagc tgccccatcc acgttgtccc 1021 tgctgggccc cactttcccc ggcttaagca gctgctccgc tgaccttaaa gacatcctga 1081 gcgaggccag caccatgcaa ctccttcagc aacagcagca ggaagcagta tccgaaggca 1141 gcagcagcgg gagagcgagg gaggcctcgg gggctcccac ttcctccaag gacaattact 1201 tagggggcac ttcgaccatt tctgacaacg ccaaggagtt gtgtaaggca gtgtcggtgt 1261 ccatgggcct gggtgtggag gcgttggagc atctgagtcc aggggaacag cttcgggggg 1321 attgcatgta cgccccactt ttgggagttc cacccgctgt gcgtcccact ccttgtgccc 1381 cattggccga atgcaaaggt tctctgctag acgacagcgc aggcaagagc actgaagata 1441 ctgctgagta ttcccctttc aagggaggtt acaccaaagg gctagaaggc gagagcctag 1501 gctgctctgg cagcgctgca gcagggagct ccgggacact tgaactgccg tctaccctgt 1561 ctctctacaa gtccggagca ctggacgagg cagctgcgta ccagagtcgc gactactaca 1621 actttccact ggctctggcc ggaccgccgc cccctccgcc gcctccccat ccccacgctc 1681 gcatcaagct ggagaacccg ctggactacg gcagcgcctg ggcggctgcg gcggcgcagt 1741 gccgctatgg ggacctggcg agcctgcatg gcgcgggtgc agcgggaccc ggttctgggt 1801 caccctcagc cgccgcttcc tcatcctggc acactctctt cacagccgaa gaaggccagt 1861 tgtatggacc gtgtggtggt ggtgggggtg gtggcggcgg cggcggcggc ggcggcggcg 1921 gcggcggcgg cggcggcggc ggcgaggcgg gagctgtagc cccctacggc tacactcggc 1981 cccctcaggg gctggcgggc caggaaagcg acttcaccgc acctgatgtg tggtaccctg 2041 gcggcatggt gagcagagtg ccctatccca gtcccacttg tgtcaaaagc gaaatgggcc 2101 cctggatgga tagctactcc ggaccttacg gggacatgcg tttggagact gccagggacc 2161 atgttttgcc cattgactat tactttccac cccagaagac ctgcctgatc tgtggagatg 2221 aagcttctgg gtgtcactat ggagctctca catgtggaag ctgcaaggtc ttcttcaaaa 2281 gagccgctga agggaaacag aagtacctgt gcgccagcag aaatgattgc actattgata 2341 aattccgaag gaaaaattgt ccatcttgtc gtcttcggaa atgttatgaa gcagggatga 2401 ctctgggagc ccggaagctg aagaaacttg gtaatctgaa actacaggag gaaggagagg 2461 cttccagcac caccagcccc actgaggaga caacccagaa gctgacagtg tcacacattg 2521 aaggctatga atgtcagccc atctttctga atgtcctgga agccattgag ccaggtgtag 2581 tgtgtgctgg acacgacaac aaccagcccg actcctttgc agccttgctc tctagcctca 2641 atgaactggg agagagacag cttgtacacg tggtcaagtg ggccaaggcc ttgcctggct 2701 tccgcaactt acacgtggac gaccagatgg ctgtcattca gtactcctgg atggggctca 2761 tggtgtttgc catgggctgg cgatccttca ccaatgtcaa ctccaggatg ctctacttcg 2821 cccctgatct ggttttcaat gagtaccgca tgcacaagtc ccggatgtac agccagtgtg 2881 tccgaatgag gcacctctct caagagtttg gatggctcca aatcaccccc caggaattcc 2941 tgtgcatgaa agcactgcta ctcttcagca ttattccagt ggatgggctg aaaaatcaaa 3001 aattctttga tgaacttcga atgaactaca tcaaggaact cgatcgtatc attgcatgca 3061 aaagaaaaaa tcccacatcc tgctcaagac gcttctacca gctcaccaag ctcctggact 3121 ccgtgcagcc tattgcgaga gagctgcatc agttcacttt tgacctgcta atcaagtcac 3181 acatggtgag cgtggacttt ccggaaatga tggcagagat catctctgtg caagtgccca 3241 agatcctttc tgggaaagtc aagcccatct atttccacac ccagtgaagc attggaaacc 3301 ctatttcccc accccagctc atgccccctt tcagatgtct tctgcctgtt ataactctgc 3361 actactcctc tgcagtgcct tggggaattt cctctattga tgtacagtct gtcatgaaca 3421 tgttcctgaa ttctatttgc tgggcttttt ttttctcttt ctctcctttc tttttcttct 3481 tccctcccta tctaaccctc ccatggcacc ttcagacttt gcttcccatt gtggctccta 3541 tctgtgtttt gaatggtgtt gtatgccttt aaatctgtga tgatcctcat atggcccagt 3601 gtcaagttgt gcttgtttac agcactactc tgtgccagcc acacaaacgt ttacttatct 3661 tatgccacgg gaagtttaga gagctaagat tatctgggga aatcaaaaca aaaacaagca 3721 aacaaaaaaa aaaagcaaaa acaaaacaaa aaataagcca aaaaaccttg ctagtgtttt 3781 ttcctcaaaa ataaataaat aaataaataa atacgtacat acatacacac atacatacaa 3841 acatatagaa atccccaaag aggccaatag tgacgagaag gtgaaaattg caggcccatg 3901 gggagttact gattttttca tctcctccct ccacgggaga ctttattttc tgccaatggc 3961 tattgccatt agagggcaga gtgaccccag agctgagttg ggcagggggg tggacagaga 4021 ggagaggaca aggagggcaa tggagcatca gtacctgccc acagccttgg tccctggggg 4081 ctagactgct caactgtgga gcaattcatt atactgaaaa tgtgcttgtt gttgaaaatt 4141 tgtctgcatg ttaatgcctc acccccaaac ccttttctct ctcactctct gcctccaact 4201 tcagattgac tttcaatagt ttttctaaga cctttgaact gaatgttctc ttcagccaaa 4261 acttggcgac ttccacagaa aagtctgacc actgagaaga aggagagcag agatttaacc 4321 ctttgtaagg ccccatttgg atccaggtct gctttctcat gtgtgagtca gggaggagct 4381 ggagccagag gagaagaaaa tgatagcttg gctgttctcc tgcttaggac actgactgaa 4441 tagttaaact ctcactgcca ctaccttttc cccaccttta aaagacctga atgaagtttt 4501 ctgccaaact ccgtgaagcc acaagcacct tatgtcctcc cttcagtgtt ttgtgggcct 4561 gaatttcatc acactgcatt tcagccatgg tcatcaagcc tgtttgcttc ttttgggcat 4621 gttcacagat tctctgttaa gagcccccac caccaagaag gttagcaggc caacagctct 4681 gacatctatc tgtagatgcc agtagtcaca aagatttctt accaactctc agatcgctgg 4741 agcccttaga caaactggaa agaaggcatc aaagggatca ggcaagctgg gcgtcttgcc 4801 cttgtccccc agagatgata ccctcccagc aagtggagaa gttctcactt ccttctttag 4861 agcagctaaa ggggctaccc agatcagggt tgaagagaaa actcaattac cagggtggga 4921 agaatgaagg cactagaacc agaaaccctg caaatgctct tcttgtcacc cagcatatcc 4981 acctgcagaa gtcatgagaa gagagaagga acaaagagga gactctgact actgaattaa 5041 aatcttcagc ggcaaagcct aaagccagat ggacaccatc tggtgagttt actcatcatc 5101 ctcctctgct gctgattctg ggctctgaca ttgcccatac tcactcagat tccccacctt 5161 tgttgctgcc tcttagtcag agggaggcca aaccattgag actttctaca gaaccatggc 5221 ttctttcgga aaggtctggt tggtgtggct ccaatacttt gccacccatg aactcagggt 5281 gtgccctggg acactggttt tatatagtct tttggcacac ctgtgttctg ttgacttcgt 5341 tcttcaagcc caagtgcaag ggaaaatgtc cacctacttt ctcatcttgg cctctgcctc 5401 cttacttagc tcttaatctc atctgttgaa ctcaagaaat caagggccag tcatcaagct 5461 gcccatttta attgattcac tctgtttgtt gagaggatag tttctgagtg acatgatatg 5521 atccacaagg gtttccttcc ctgatttctg cattgatatt aatagccaaa cgaacttcaa 5581 aacagcttta aataacaagg gagaggggaa cctaagatga gtaatatgcc aatccaagac 5641 tgctggagaa aactaaagct gacaggttcc ctttttgggg tgggatagac atgttctggt 5701 tttctttatt attacacaat ctggctcatg tacaggatca cttttagctg ttttaaacag 5761 aaaaaaatat ccaccactct tttcagttac actaggttac attttaatag gtcctttaca 5821 tctgttttgg aatgattttc atcttttgtg atacacagat tgaattatat cattttcata 5881 tctctccttg taaatactag aagctctcct ttacatttct ctatcaaatt tttcatcttt 5941 atgggtttcc caattgtgac tcttgtcttc atgaatatat gtttttcatt tgcaaaagcc 6001 aaaaatcagt gaaacagcag tgtaattaaa agcaacaact ggattactcc aaatttccaa 6061 atgacaaaac tagggaaaaa tagcctacac aagcctttag gcctactctt tctgtgcttg 6121 ggtttgagtg aacaaaggag attttagctt ggctctgttc tcccatggat gaaaggagga 6181 ggattttttt tttcttttgg ccattgatgt tctagccaat gtaattgaca gaagtctcat 6241 tttgcatgcg ctctgctcta caaacagagt tggtatggtt ggtatactgt actcacctgt 6301 gagggactgg ccactcagac ccacttagct ggtgagctag aagatgagga tcactcactg 6361 gaaaagtcac aaggaccatc tccaaacaag ttggcagtgc tcgatgtgga cgaagagtga 6421 ggaagagaaa aagaaggagc accagggaga aggctccgtc tgtgctgggc agcagacagc 6481 tgccaggatc acgaactctg tagtcaaaga aaagagtcgt gtggcagttt cagctctcgt 6541 tcattgggca gctcgcctag gcccagcctc tgagctgaca tgggagttgt tggattcttt 6601 gtttcatagc tttttctatg ccataggcaa tattgttgtt cttggaaagt ttattatttt 6661 tttaactccc ttactctgag aaagggatat tttgaaggac tgtcatatat ctttgaaaaa 6721 agaaaatctg taatacatat atttttatgt atgttcactg gcactaaaaa atatagagag 6781 cttcattctg tcctttgggt agttgctgag gtaattgtcc aggttgaaaa ataatgtgct 6841 gatgctagag tccctctctg tccatactct acttctaaat acatataggc atacatagca 6901 agttttattt gacttgtact ttaagagaaa atatgtccac catccacatg atgcacaaat 6961 gagctaacat tgagcttcaa gtagcttcta agtgtttgtt tcattaggca cagcacagat 7021 gtggcctttc cccccttctc tcccttgata tctggcaggg cataaaggcc caggccactt 7081 cctctgcccc ttcccagccc tgcaccaaag ctgcatttca ggagactctc tccagacagc 7141 ccagtaacta cccgagcatg gcccctgcat agccctggaa aaataagagg ctgactgtct 7201 acgaattatc ttgtgccagt tgcccaggtg agagggcact gggccaaggg agtggttttc 7261 atgtttgacc cactacaagg ggtcatggga atcaggaatg ccaaagcacc agatcaaatc 7321 caaaacttaa agtcaaaata agccattcag catgttcagt ttcttggaaa aggaagtttc 7381 tacccctgat gcctttgtag gcagatctgt tctcaccatt aatctttttg aaaatctttt 7441 aaagcagttt ttaaaaagag agatgaaagc atcacattat ataaccaaag attacattgt 7501 acctgctaag ataccaaaat tcataagggc agggggggag caagcattag tgcctctttg 7561 ataagctgtc caaagacaga ctaaaggact ctgctggtga ctgacttata agagctttgt 7621 gggttttttt ttccctaata atatacatgt ttagaagaat tgaaaataat ttcgggaaaa 7681 tgggattatg ggtccttcac taagtgattt tataagcaga actggctttc cttttctcta 7741 gtagttgctg agcaaattgt tgaagctcca tcattgcatg gttggaaatg gagctgttct 7801 tagccactgt gtttgctagt gcccatgtta gcttatctga agatgtgaaa cccttgctga 7861 taagggagca tttaaagtac tagattttgc actagaggga cagcaggcag aaatccttat 7921 ttctgcccac tttggatggc acaaaaagtt atctgcagtt gaaggcagaa agttgaaata 7981 cattgtaaat gaatatttgt atccatgttt caaaattgaa atatatatat atatatatat 8041 atatatatat atatatatat agtgtgtgtg tgtgttctga tagctttaac tttctctgca 8101 tctttatatt tggttccaga tcacacctga tgccatgtac ttgtgagaga ggatgcagtt 8161 ttgttttgga agctctctca gaacaaacaa gacacctgga ttgatcagtt aactaaaagt 8221 tttctcccct attgggtttg acccacaggt cctgtgaagg agcagaggga taaaaagagt 8281 agaggacatg atacattgta ctttactagt tcaagacaga tgaatgtgga aagcataaaa 8341 actcaatgga actgactgag atttaccaca gggaaggccc aaacttgggg ccaaaagcct 8401 acccaagtga ttgaccagtg gccccctaat gggacctgag ctgttggaag aagagaactg 8461 ttccttggtc ttcaccatcc ttgtgagaga agggcagttt cctgcattgg aacctggagc 8521 aagcgctcta tctttcacac aaattccctc acctgagatt gaggtgctct tgttactggg 8581 tgtctgtgtg ctgtaattct ggttttggat atgttctgta aagattttga caaatgaaaa 8641 tgtgtttttc tctgttaaaa cttgtcagag tactagaagt tgtatctctg taggtgcagg 8701 tccatttctg cccacaggta gggtgttttt ctttgattaa gagattgaca cttctgttgc 8761 ctaggacctc ccaactcaac catttctagg tgaaggcaga aaaatccaca ttagttactc 8821 ctcttcagac atttcagctg agataacaaa tcttttggaa ttttttcacc catagaaaga 8881 gtggtagata tttgaattta gcaggtggag tttcatagta aaaacagctt ttgactcagc 8941 tttgatttat cctcatttga tttggccaga aagtaggtaa tatgcattga ttggcttctg 9001 attccaattc agtatagcaa ggtgctaggt tttttccttt ccccacctgt ctcttagcct 9061 ggggaattaa atgagaagcc ttagaatggg tggcccttgt gacctgaaac acttcccaca 9121 taagctactt aacaagattg tcatggagct gcagattcca ttgcccacca aagactagaa 9181 cacacacata tccatacacc aaaggaaaga caattctgaa atgctgtttc tctggtggtt 9241 ccctctctgg ctgctgcctc acagtatggg aacctgtact ctgcagaggt gacaggccag 9301 atttgcatta tctcacaacc ttagcccttg gtgctaactg tcctacagtg aagtgcctgg 9361 ggggttgtcc tatcccataa gccacttgga tgctgacagc agccaccatc agaatgaccc 9421 acgcaaaaaa aagaaaaaaa aaattaaaaa gtcccctcac aacccagtga cacctttctg 9481 ctttcctcta gactggaaca ttgattaggg agtgcctcag acatgacatt cttgtgctgt 9541 ccttggaatt aatctggcag caggagggag cagactatgt aaacagagat aaaaattaat

9601 tttcaatatt gaaggaaaaa agaaataaga agagagagag aaagaaagca tcacacaaag

9661 attttcttaa aagaaacaat tttgcttgaa atctctttag atggggctca tttctcacgg

9721 tggcacttgg cctccactgg gcagcaggac cagctccaag cgctagtgtt ctgttctctt

9781 tttgtaatct tggaatcttt tgttgctcta aatacaatta aaaatggcag aaacttgttt

9841 gttggactac atgtgtgact ttgggtctgt ctctgcctct gctttcagaa atgtcatcca

9901 ttgtgtaaaa tattggctta ctggtctgcc agctaaaact tggccacatc ccctgttatg

9961 gctgcaggat cgagttattg ttaacaaaga gacccaagaa aagctgctaa tgtcctctta

10021 tcattgttgt taatttgtta aaacataaag aaatctaaaa tttcaaaaaa

By "Androgen Receptor (AR)" is meant a polypeptide or fragment thereof having at least about 85% amino acid identity to NCBI Accession No. AAA51771, version AAA51771.1, incorporated herein by reference, as reproduced below (SEQ ID NO: 47):

1 mevqlglgrv yprppsktyr gafqnlfqsv reviqnpgpr hpeaasaapp gasllllqqq

61 qqqqqqqqqq qqqqqqqets prqqqqqqge dgspqahrrg ptgylvldee qqpsqpqsal

121 echpergcvp epgaavaask glpqqlpapp deddsaapst lsllgptfpg lsscsadlkd

181 ilseastmql lqqqqqeavs egsssgrare rsgaptsskd nylggtstis dnakelckav

241 svsmglgvea lehlspgeql rgdcmyapll gvppavrptp caplaeckgs llddsagkst

301 edtaeyspfk ggytkglege slgcsgsaaa gssgtlelps tlslyksgal deaaayqsrd

361 yynfplalag pppppppphp hariklenpl dygsawaaaa aqcrygdlas lhgagaagpg

421 sgspsaaass swhtlftaee gqlygpcggg gggggggggg gggggggggg eagavapygy

481 trppqglagq esdftapdvw ypggmvsrvp ypsptcvkse mgpwmdsysg pygdmrleta

541 rdhvlpidyy fppqktclic gdeasgchyg altcgsckvf fkraaegkqk ylcasrndct

601 idkfrrkncp scrlrkcyea gmtlgarklk klgnlklqee geassttspt eettqkltvs

661 hiegyecqpi flnvleaiep g vcaghdnn qpds faalls slnelgerql vh vkwakal

721 pgfrnlhvdd qmaviqyswm glmvfamgwr sftnvnsrml yfapdlvfne yrmhksrmys

781 qcvrmrhlsq efgwlqitpq eflcmkalll fsiipvdglk nqkffdelrm nyikeldrii

841 ackrknptsc srrfyqltkl ldsvqpiare lhqftfdlli kshmvs dfp emmaeiisvq

901 vpkilsgkvk piyfhtq

The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Those of ordinary skill in the art may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for describing particular embodiments only and is not intended to be limiting of the disclosure. All publications, patent applications, patents, figures and other references mentioned herein are expressly incorporated by reference in their entirety.

The practice of the present subject matter may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, molecular biology (including recombinant techniques), cell biology, and biochemistry, which are within the skill of the art. Such conventional techniques include, but are not limited to, preparation of synthetic

polynucleotides, polymerization techniques, chemical and physical analysis of polymer particles, preparation of nucleic acid libraries, nucleic acid sequencing and analysis, and the like. Specific illustrations of suitable techniques can be used by reference to the examples provided herein. Other equivalent conventional procedures can also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Genome Analysis: A

Laboratory Manual Series (Vols. I-IV), PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Hermanson, Bioconjugate Techniques, Second Edition (Academic Press, 2008); Merkus, Particle Size Measurements (Springer, 2009); Rubinstein and Colby, Polymer Physics (Oxford University Press, 2003); "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (Gait, ed., 1984); "Animal Cell Culture" (Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental

Immunology" (Wei & Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (Miller & Calos, eds., 1987); "Current Protocols in Molecular Biology" (Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology" (Coligan et al., eds., 1991). These techniques are applicable to the production of the polynucleotides and polypeptides, and, as such, can be considered in making and practicing the disclosure.

The primers of the disclosure and their functional derivatives can include any suitable polynucleotide that can hybridize to a target sequence of interest. The primers can serve to prime nucleic acid synthesis, e.g., in a PCR reaction. Typically, the primer functions as a substrate onto which nucleotides can be polymerized by a polymerase; in some embodiments, however, the primer can become incorporated into the synthesized nucleic acid strand and provide a site to which another primer can hybridize to prime synthesis of a new strand that is complementary to the synthesized nucleic acid molecule. The primers of the disclosure may be comprised of any combination of nucleotides or analogs thereof, which may be optionally linked to form a linear polymer of any suitable length. In some embodiments, the primers are single- stranded oligonucleotides or polynucleotides. In some embodiments, the primers are single- stranded. The primers can also be double-stranded. The primers optionally occur naturally, as in a purified restriction digest, or can be produced synthetically. In some embodiments, the primers act as a point of initiation for amplification or synthesis when exposed to amplification or synthesis conditions; such amplification or synthesis can occur in a template-dependent fashion and optionally results in formation of a primer extension product that is complementary to at least a portion of the target sequence.

Exemplary amplification or synthesis conditions can include contacting the primer with a polynucleotide template (e.g., a template including a target SLGI sequence or sequences), nucleotides and an inducing agent such as a polymerase at a suitable temperature and pH to induce polymerization of nucleotides onto an end of the target-specific primer. If double- stranded, the primer can optionally be treated to separate its strands before being used to prepare primer extension products. In some embodiments, the primer is an oligodeoxyribonucleotide or an oligoribonucleotide. In some embodiments, the primer can include one or more nucleotide analogs. The exact length and/or composition, including sequence, of the target-specific primer can influence many properties, including melting temperature (Tm), GC content, formation of secondary structures, repeat nucleotide motifs, length of predicted primer extension products, extent of coverage across a nucleic acid molecule of interest, number of primers present in a single amplification or synthesis reaction, presence of nucleotide analogs or modified nucleotides within the primers, and the like.

In some embodiments, a primer can be paired with a compatible primer within an amplification or synthesis reaction to form a primer pair consisting or a forward primer and a reverse primer. In some embodiments, the forward primer of the primer pair includes a sequence that is substantially complementary to at least a portion of a strand of a nucleic acid molecule, and the reverse primer of the primer of the primer pair includes a sequence that is substantially identical to at least of portion of the strand. In some embodiments, the forward primer and the reverse primer are capable of hybridizing to opposite strands of a nucleic acid duplex.

Optionally, the forward primer primes synthesis of a first nucleic acid strand, and the reverse primer primes synthesis of a second nucleic acid strand, wherein the first and second strands are substantially complementary to each other, or can hybridize to form a double-stranded nucleic acid molecule.

In some embodiments, one end of an amplification or synthesis product is defined by the forward primer and the other end of the amplification or synthesis product is defined by the reverse primer. In some embodiments, where the amplification or synthesis of lengthy primer extension products is required, such as amplifying an exon, coding region, or gene, several primer pairs can be created than span the desired length to enable sufficient amplification of the region. In some embodiments, a primer can include one or more cleavable groups.

In some embodiments, primer lengths are in the range of about 10 to about 60

nucleotides, about 12 to about 50 nucleotides and about 15 to about 40 nucleotides in length. Typically, a primer is capable of hybridizing to a corresponding target sequence and undergoing primer extension when exposed to amplification conditions in the presence of dNTPs and a polymerase. In some instances, the particular nucleotide sequence or a portion of the primer is known at the outset of the amplification reaction or can be determined by one or more of the methods disclosed herein. In some embodiments, the primer includes one or more cleavable groups at one or more locations within the primer.

In the various disclosed embodiments, any suitable length primers are contemplated. The length of the primers may be limited by a minimum primer length threshold and a maximum primer length, and a length score for the primers may be set so as to decrease as the length gets shorter than the minimum primer length threshold and to decrease as the length gets longer than the maximum primer length threshold. In an embodiment, the minimum primer length threshold may be 16. In other embodiments, the minimum primer length threshold may be 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5, for example, and may also be 17, 18, 19, 20, 21, 22, 23, and 24, for example. In an embodiment, the maximum primer length threshold may be 28. In other embodiments, the maximum primer length threshold may be 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, for example, and may also be 27, 26, 25, 24, 23, 22, 21, and 20, for example. In an embodiment, the primer length criterion may be given a score of 1.0 if the length thresholds are satisfied, for example, and that score may go down to 0.0 as the primer length diverges from the minimum or maximum length threshold. For example, if the maximum primer length threshold were set to 28, then the score could be set to 1.0 if the length does not exceed 28, to 0.7 if the length is 29, to 0.6 if the length is 30, to 0.5 if the length is 31, to 0.3 if the length is 32, to 0.1 if the length is 33, and to 0.0 if the length is 34 or more. The attribute/score could be scaled between values other than 0.0 and 1.0, of course, and the function defining how the score varies with an increase difference relative to the threshold could be any other or more complex linear or non-linear function that does not lead to increases in score for primer that further diverge from length thresholds.

In various embodiments, the method of the disclosure preferably utilizes wildtype primer sets that are modified to prevent their extension by a polymerase in a PCR reaction or in a PCR- based assay. Such modification can be any known in the art. For example, the wildtype primers can be modified with a 3' end blocking group which prevents extension by DNA polymerase. One such blocking group can include a 3 '-end dideoxyCytosine (ddC), which is covalently modified on the 3' terminal phosphate and prevents extension by DNA polymerase. Any other suitable blocking group known in the art is contemplated which blocks DNA polymerase extension.

In various embodiments, the detection of PCR products resulting from the methods of the disclosure may be performed by any known read-out methodology, such as by nucleotide sequence, gel-based detection, or by molecular reporter system. Such read-out methodologies are well-known in the art and the skilled person will understand how to use such read-out techniques to in the disclosed detection methods.

In various aspects, the read-out methods may be conducted with the aid of a computer- based system configured to execute machine-readable instructions, which, when executed by a processor of the system causes the system to perform steps including determining the identity, size, nucleotide sequence or other measurable characteristics of the amplicons produced in the method of the disclosure. One or more features of any one or more of the above-discussed teachings and/or exemplary embodiments may be performed or implemented using appropriately configured and/or programmed hardware and/or software elements. Determining whether an embodiment is implemented using hardware and/or software elements may be based on any number of factors, such as desired computational rate, power levels, heat tolerances, processing cycle budget, input data rates, output data rates, memory resources, data bus speeds, etc., and other design or performance constraints.

Examples of hardware elements may include control units, processors, microprocessors, input(s) and/or output(s) (I/O) device(s) (or peripherals) that are communicatively coupled via a local interface circuit, circuit elements (e.g., transistors, resistors, capacitors, inductors, and so forth), integrated circuits, application specific integrated circuits (ASIC), programmable logic devices (PLD), digital signal processors (DSP), field programmable gate array (FPGA), logic gates, registers, semiconductor device, chips, microchips, chip sets, and so forth. The local interface may include, for example, one or more buses or other wired or wireless connections, controllers, buffers (caches), drivers, repeaters and receivers, etc., to allow appropriate communications between hardware components. A processor is a hardware device for executing software, particularly software stored in memory. The processor can be any custom made or commercially available processor, a central processing unit (CPU), an auxiliary processor among several processors associated with the computer, a semiconductor-based microprocessor (e.g., in the form of a microchip or chip set), a macroprocessor, or generally any device for executing software instructions. A processor can also represent a distributed processing architecture. The I/O devices can include input devices, for example, a keyboard, a mouse, a scanner, a microphone, a touch screen, an interface for various medical devices and/or laboratory instruments, a bar code reader, a stylus, a laser reader, a radio-frequency device reader, etc. Furthermore, the I/O devices also can include output devices, for example, a printer, a bar code printer, a display, etc. Finally, the I/O devices further can include devices that communicate as both inputs and outputs, for example, a modulator/demodulator (modem; for accessing another device, system, or network), a radio frequency (RF) or other transceiver, a telephonic interface, a bridge, a router, etc. It is expressly contemplated that the components and/or elements described herein can be implemented as software being stored on a tangible (non-transitory) computer- readable medium (e.g., disks/CDs/etc.) having program instructions executing on a computer, hardware, firmware, or a combination thereof.

Examples of software may include software components, programs, applications, computer programs, application programs, system programs, machine programs, operating system software, middleware, firmware, software modules, routines, subroutines, functions, methods, procedures, software interfaces, application program interfaces (API), instruction sets, computing code, computer code, code segments, computer code segments, words, values, symbols, or any combination thereof. A software in memory may include one or more separate programs, which may include ordered listings of executable instructions for implementing logical functions. The software in memory may include a system for identifying data streams in accordance with the present teachings and any suitable custom made or commercially available operating system (O/S), which may control the execution of other computer programs such as the system, and provides scheduling, input-output control, file and data management, memory management, communication control, etc.

According to various exemplary embodiments, one or more features of any one or more of the above-discussed teachings and/or exemplary embodiments may be performed or implemented at least partly using a distributed, clustered, remote, or cloud computing resource.

According to various exemplary embodiments, one or more features of any one or more of the above-discussed teachings and/or exemplary embodiments may be performed or implemented using a source program, executable program (object code), script, or any other entity comprising a set of instructions to be performed. When using a source program, the program can be translated via a compiler, assembler, interpreter, etc., which may or may not be included within the memory, so as to operate properly in connection with the O/S. The instructions may be written using (a) an object-oriented programming language, which has classes of data and methods, or (b) a procedural programming language, which has routines, subroutines, and/or functions, which may include, for example, C, C++, Pascal, Basic, Fortran, Cobol, Pert, Java, and Ada.

According to various exemplary embodiments, one or more of the above-discussed exemplary embodiments may include transmitting, displaying, storing, printing or outputting to a user interface device, a computer readable storage medium, a local computer system or a remote computer system, information related to any information, signal, data, and/or intermediate or final results that may have been generated, accessed, or used by such exemplary embodiments. Such transmitted, displayed, stored, printed or outputted information can take the form of searchable and/or filterable lists of runs and reports, pictures, tables, charts, graphs, spreadsheets, correlations, sequences, and combinations thereof, for example.

Various additional exemplary embodiments may be derived by repeating, adding, or substituting any generically or specifically described features and/or components and/or substances and/or steps and/or operating conditions set forth in one or more of the above- described exemplary embodiments. Further, it should be understood that an order of steps or order for performing certain actions is immaterial so long as the objective of the steps or action remains achievable, unless specifically stated otherwise. Furthermore, two or more steps or actions can be conducted simultaneously so long as the objective of the steps or action remains achievable, unless specifically stated otherwise. Moreover, any one or more feature, component, aspect, step, or other characteristic mentioned in one of the above-discussed exemplary embodiments may be considered to be a potential optional feature, component, aspect, step, or other characteristic of any other of the above-discussed exemplary embodiments so long as the objective of such any other of the above-discussed exemplary embodiments remains achievable, unless specifically stated otherwise.

The term "cancer," as used herein, may include, but is not limited to: biliary tract cancer; bladder cancer; brain cancer including glioblastomas and medulloblastomas; breast cancer;

cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic and myelogenous leukemia;

multiple myeloma; AIDS -associated leukemia and adult T-cell leukemia lymphoma;

intraepithelial neoplasms including Bowen's disease and Paget' s disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastomas; oral cancer including squamous cell carcinoma; ovarian cancer including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer including melanoma, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma, teratomas, choriocarcinomas; stromal tumors and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullar carcinoma; and renal cancer including adenocarcinoma and Wilms' tumor. Commonly encountered cancers include breast, prostate, lung, ovarian, colorectal, and brain cancer. In general, an effective amount of the compositions of the disclosure for treating cancer will be that amount necessary to inhibit mammalian cancer cell proliferation in situ. Those of ordinary skill in the art are well-schooled in the art of evaluating effective amounts of anti -cancer agents.

Reference will now be made in detail to exemplary embodiments of the disclosure. While the disclosure will be described in conjunction with the exemplary embodiments, it will be understood that it is not intended to limit the disclosure to those embodiments. To the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the disclosure as defined by the appended claims. EXAMPLES

The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The contents of all references, GenBank Accession and Gene numbers, and published patents and patent applications cited throughout the application are hereby incorporated by reference. Those skilled in the art will recognize that the disclosure may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the scope of the disclosure.

According to the techniques herein, the simultaneous lentiviral delivery of paired guide RNAs (pgRNAs) targeting two separate genes in a CRISPR/Cas9 knockout (KO) screen may provide a cost-effective approach for high throughput identification of SLGIs. The present disclosure provides experimental technologies and computational methods to conduct large-scale prediction, identification, and validation of synthetic lethal gene interaction (SLGIs) involved in cancer. In particular, the below Examples describe a novel pgRNA CRISPR vector system, vector library, screening techniques and integrative algorithms to find novel therapies targeting cancers with tumor suppressor gene (TSG) loss. Prior art SLGI studies in humans have either focused on a single SLGI pair or compared essential genes between cancer cell lines where one anchor gene is wild-type or mutant (e.g., a "1 x n" design) or via combinatorial pairs (e.g., an "a x b" design), which drastically limits the number of effective SLGI pairs that can be investigated. Due to these limitations, the current collection of human SLGI pairs that have a high degree of confidence is only about 100. The present disclosure provides cutting-edge and cost-effective technologies for high throughput identification, prediction, and validation of SLGIs in individual cell lines. First, the techniques herein provide a novel pooled CRISPR/Cas9 double KO screening technique in which each lentivirus carries pgRNAs designed to simultaneously KO specific pairs of SLGI partners. Second, the techniques herein provide a novel computational algorithm that integrates pgRNA screening data, available single guide RNA (sgRNA) CRISPR screening data, and The Cancer Genome Atlas (TCGA) tumor profiling data, to predict SLGI pairs. Third, the techniques herein provide large-scale pgRNA CRISPR screens across different cancer cell lines to identify and characterize cancer-specific SLGIs. The techniques herein will enable comprehensive identification of therapeutic targets for cancers with TSG loss, and will inform better development of precision cancer medicine. Example 1 : CRISPR Screens with a "1 x n" Design Identified P21 (RAC1) Activated Kinase 2 (PAK2) as a C-Src Tyrosine Kinase (CSK) SLGI Partner in Breast Cancers

CRISPR/Cas9 KO libraries with a sgRNA per vector targeting exons have been proven to be a powerful genetic screen platform (see e.g., reference 7). The techniques herein expand sgRNA screening to a pgRNA modality. As shown in FIGS. 2A-2F, initial experiments have shown that two rounds of CRISPR screening using a "1 x n" design identified a unique synthetic lethal pair that drives hormone independent cell growth in breast cancer models. In particular, these CRISPR screens identified PAK2 and CSK as a SLGI pair in breast cancer cells.

As shown in FIG. 2A, a genome-wide sgRNA CRISPR knockout screen was first conducted in the T47D and MCF7 breast cancer cell lines to search for key genes whose loss would specifically drive estrogen-independent growth. CSK was identified as the strongest positively-selected hit in both T47D and MCF7 cell lines (FIGS. 2A-C). CSK knockout confers hormone independent growth, which could be fully reversed by the overexpression of a human CSK cDNA (FIG. 2D).

To identify key genes that drive hormone independent growth upon CSK loss, a second round of genome-wide CRISPR screen was performed to compare the T47D-CSK null vs T47D- CSK wild type cells (FIG. 2E). This secondary screen identified PAK2 as possibly having a SLGI in combination with CSK because PAK2 is uniquely essential in the CSK-null cells (FIG. 2F). Based on this method, a series of genome-wide CRISPR screens were conducted by simultaneously knocking out another positively-selected gene(s) such as Tuberous Sclerosis 1/2 (TSCl/2) in T47D, which provides multiple "1 x n" design SLGI pairs with which to train the algorithms described below.

Example 2: A pgRNA Library Enables CRISPR Deletion Screens to Find Functional IncRNAs in Human Cancers

The simultaneous expression of two gRNAs targeting two different genes in the genome may introduce indels to KO both genes. Alternatively, if the two targeting sites are close to each other, the fragment in between could be deleted (see e.g., reference 26). Therefore, with a reliable cloning method to construct pgRNA CRISPR libraries, a high-throughput SLGI screen(s) or deletion screen(s) may be conducted. A two-step pgRNA library (see e.g., reference 27) was capable of delivering the expression of two gRNAs per lentiviral vector and building the cell library pool in a similar way as in single gene CRISPR KO libraries (FIGS. 3 A-3B) and screening methods (FIG. 3C) as described in Zhu et al. (Nat Biotechnol. 2016 Dec;34(12): 1279-1286). FIG. 3B shows DNA sequences of the engineered oligo and linker between the two gRNAs of each pair, which sequence is set forth below (SEQ ID NO: 29):

5 ' -ATCTTGTGGAAAGGACGAAAC ACCG

[+guidel+]

GTTTAGAGACGAGCCTCTATACTTACTAAACGTGATCGTCTCAACCG

[+guide2+]

GTTTAAGAGCTATGCTGGAAACAGC-3 '

In this screen, the same U6 was used in front of both gRNAs; therefore, it was only possible to sequence the first gRNA as a barcode for each pgRNA pair and decode the screen results. Unfortunately, this sequencing strategy could not assay whether the pairs swapped during the library construction, screening, or sequencing preparation processes because it only decodes the first half of pgRNA information. Additionally, this strategy also limits the choices of pgRNA design by requiring the first gRNA to be unique in every pair. This screening strategy also suffered a relatively high false negative rate, potentially due to PCR

swapping/recombination that disrupting the designed pgRNA pairing.

Example 3 : Novel pgRNA Oligo Design with a Unique Linker Improves the Quality of the pgRNA Library

According to the techniques herein, paired-end sequencing could decode both pgRNAs in each pair and reveal a substantial portion of the swapped pairs in the library. To reduce the swapping rate, the present disclosure provides a novel pgRNA expression system design in which two different U6 promoters (e.g., a human U6 promoter and mouse U6 promoter) are used to drive expression of two gRNAs, each of which is followed sequentially by a different scaffold sequence that includes a tracrRNA sequence. Advantageously, this design minimizes the possibility of lentiviral replication-generated recombination (see e.g., references 28 and 29), and it decreases the swapping rate at the cell library level.

As shown in FIG. 1 and FIG. 3H, paired-end sequencing analysis of swapped pairs generated in prior art pgRNA library design revealed that the first amplification step of the oligo library may generate around 50% of all swapped pairs in the library, and also that these swapped pairs are preserved in later plasmid vector and cell libraries. It was believed that the common linker between the two gRNAs resulted in the PCR-generated swapping events. In a pilot 7.5K pgRNA library construction experiment in which two gRNAs flank a cis-element for deletion, this hypothesis was confirmed when an altered oligo design in which every pair contains a unique linker completely eliminated the swapping issue during the first PCR step. However, in the second cloning step, the tracrRNA-U6 promoter sequence is inserted between the first gRNA sequence and the second gRNA sequence, and the inserted tracrRNA-U6 fragment then becomes a common linker. As shown in FIG. 31, the analysis of the colony PCR amplicons from the complete vector library, in which the PCR-related recombination events are eliminated because each colony has only one pgRNA vector, 12/12 of the pgRNAs are correct pairs.

To prepare the deep sequencing samples from the vector pool or the genomic DNA of the cell library, it was necessary to PCR amplify the pgRNA sequence and add sequencing adaptors, which again created swapped/recombined pairs at a frequency of about 50%. However, since the screening was still done with the correct pairing and swapping only happened during the final step of preparing the library before sequencing, it was possible to filter the pgRNAs with the wrong pairing from the sequencing data. A pilot 7.5K library screen yielded good results with low false discovery rate even with a single replicate, demonstrating the ability of the techniques herein to conduct robust and cost-effective pgRNA CRISPR screens in cancer cell lines.

The techniques herein provide, in part, a pgRNA library vector including two gRNA cassettes and a Cas9 expression cassette (see e.g., FIG. 3D) and methods for constructing the same (FIG. 3E). In the library vector design, two different U6 promoters (e.g., a human U6 promoter and mouse U6 promoter) may be used to drive expression of two different gRNAs in conjunction with two different gRNA scaffolds. It is contemplated with the scope of the disclosure that any of a variety of different promoters may be used, and one of skill in the art will appreciate that the choice of promoters may vary depending upon a variety of factors such as the cell type and/or disease state of the cell line that is being screened. For example, alternate promoters may include, but are not limited to, the HI promoter (see e.g., Myslinski, E., Ame, J.C., Krol, A. and Carbon, P. (2001) An unusually compact external promoter for RNA polymerase III transcription of the human HIRNA gene. Nucleic Acids Res., 29, 2502-2509), the 7SK promoter (see e.g., Murphy, S., Di Liegro, C. and Melli, M. (1987) The in vitro transcription of the 7SK RNA gene by RNA polymerase III is dependent only on the presence of an upstream promoter. Cell, 51, 81-87), or a modified bovine U6 promoter (see e.g., Adamson et al. (2016) A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic

Dissection of the Unfolded Protein Response. Cell. Volume 167, Issue 7, pl867-1882).

Library Construction: Design and synthesis of the oligo library

FIG. 3E shows a method of making the present pgRNA vector that greatly reduces, or eliminates, internal recombination between pgRNAs, thereby increasing the fidelity of resulting pgRNA libraries.

In an exemplary embodiment shown in FIG. 3F, the design of the oligo may be as follows (SEQ ID NO: 16): 5'-

GTGGAAAGGACGAAAC ACCG+guide 1 +GTTTNGAGACGNNNNNNNNNNNNNNNNCG TCTCNGTTG+guide2+GTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGG-3 ' . It is contemplated within the scope of the invention that each gRNA pair may have a different linker (e.g., a unique linker that may be randomly designed and assigned to a given gRNA pair), in sharp contrast to prior art methods. In this regards, the specific linker used for a given gRNA pair does not matter so long as each gRNA pair has a different linker.

While the above exemplary embodiment discloses a 16 nucleotide linker

(NNNNNNNNNNNNNNNN (SEQ ID NO: 17)), it is contemplated within the scope of the disclosure that the linker may range from 10-30 nucleotides in length. In exemplary

embodiments, the GC content of the linker may be less than or equal to 40% (e.g., 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%).

Exemplary gRNAs may be selected from any genomic regions of interest that match the PAM requirement (e.g., a trailing or leading NGG) and/or the guide efficiency model. In an exemplary embodiment, the length of both gRNAs may be 19 nucleotides, so the total length of the product is 130 nucleotides. One of skill in the art will appreciate that the length of the gRNA may be slightly longer or shorter (e.g., the gRNA length may range from about 17-27 nucleotides in length).

The manufacture of the oligo pool may be conducted by Agilent Technologies Inc. or Twist Biosciences, Inc.

An exemplary forward oligo (e.g., oligo F) may have the following sequence (SEQ ID NO: 18):

TAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACA CCG

An exemplary reverse oligo (e.g., oligo R) may have the following sequence (SEQ ID NO: 19):

ACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAA AC

Library Construction: Two-step cloning of the oligo pool into a lentiCRISPRv2 vector

FIG. 3E depicts an exemplary two step cloning process that may be used to make the library vectors disclosed herein. In a first step, a Gibson assembly reaction may be applied to an exemplary linearized (e.g., enzymatically digested) lentiCRISPRv2 vector backbone (e.g., including in 5' to 3' order: a human U6 promoter, a vector linker, and a second gRNA scaffold; see e.g., FIG. 3E, top panel) in which the vector linker has been removed and an amplified oligonucleotide library having the general structure, in 5' to 3' order, first gRNA-unique linker- second gRNA (FIG. 3F), to create an intermediate nucleic acid sequence having the following exemplary structure in 5' to 3' order: a human U6 promoter, first gRNA, a unique linker (e.g., randomized linker), second gRNA, second gRNA scaffold (see e.g., FIG. 3E, middle panel).

In an exemplary embodiment, the vector linker may have the following sequence (SEQ ID NO: 20):

GAGACGGTTGTAAATGAGCACACAAAATACACATGCTAAAATATTATATTCTATGAC CTTTATAAAATCAACCAAAATCTTCTTTTTAATAACTTTAGTATCAATAATTAGAATT TTTATGTTCCTTTTTGCAAACTTTTAATAAAAATGAGCAAAATAAAAAAACGCTAGT TTTAGTAACTCGCGTTGTTTTCTTCACCTTTAATAATAGCTACTCCACCACTTGTTCCT AAGCGGTCAGCTCCTGCTTCAATCATTTTTTGAGCATCTTCAAATGTTCTAACTCCAC CAGCTGCTTTAACTAAAGCATTGTCTTTAACAACTGACTTCATTAGTTTAACATCTTC AAATGTTGCACCTGATTTTGAAAATCCTGTTGATGTTTTAACAAATTCTAATCCAGCT TCAACAGCTATTTCACAAGCTTTCATGATTTCTTCTTTTGTTAATAAACAATTTTCCA

TAATACATTTAACAACATGTGATCCAGCTGCTTTTTTTACAGCTTTCATGTCTTCTAA

AACTAATTCATAATTTTTGTCTTTTAATGCACCAATATTTAATACCATATCAATTTCT

GTTGCACCATCTTTAATTGCTTCAGAAACTTCGAATGCTTTTGTAGCTGTTGTGCATG

CACCTAGAGGAAAACCTACAACATTTGTTATTCCTACATTTGTGCCTTTTAATAATTC

TTTACAATAGCTTGTTCAATATGAATTAACACAAACTGTTGCAAAATCAAATTCAAT

TGCTTCATCACATAATTGTTTAATTTCAGCTTTCGTAGCATCTTGTTTTAATAATGTGT

GATCTATATATTTGTTTAGTTTCATTTTTTCTCCTATATATTCATTTTTAATTTTAATTC

TTTAATAATTTCGTCTACTTTAACTTTAGCGTTTTGAACAGATTCACCAACACCTATA

AAATAAATTTTTAGTTTAGGTTCAGTTCCACTTGGGCGAACAGCAAATCATGACTTA

TCTTCTAAATAAAATTTTAGTAAGTCTTGTCCTGGCATATTATACATTCCATCGATGT

AGTCTTCAACATTAACAACTTTAAGTCCAGCAATTTGAGTTAAGGGTGTTGCTCTCA

ATGATTTCATTAATGGTTCAATTTTTAATTTCTTTTCTTCTGGTTTAAAATTCAAGTTT

AAAGTGAAAGTGTAATATGCACCCATTTCTTTAAATAAATCTTCTAAATAGTCTACT

AATGTTTTATTTTGTTTTTTATAAAATCAAGCAGCCTCTGCTATTAATATAGAAGCTT

GTATTCCATCTTTATCTCTAGCTGAGTCATCAATTACATATCCATAACTTTCTTCATA

AGCAAAAACAAAATTTAATCCGTTATCTTCTTCTTTAGCAATTTCTCTACCCATTCAT

TTAAATCCAGTTAAAGTTTTTACAATATTAACTCCATATTTTTCATGAGCGATTCTAT

CACCCAAATCACTTGTTACAAAACTTGAATATAGAGCCGGATTTTTTGGAATGCTAT

TTAAGCGTTTTAGATTTGATAATTTTCAATCAATTAAAATTGGTCCTGTTTGATTTCC

ATCTAATCTTACAAAATGACCATCATGTTTTATTGCCATTCCAAATCTGTCAGCATCT

GGGTCATTCATAATAATAATATCTGCATCATGTTTAATACCATATTCAAGCGGTATTT

TTCATGCAGGATCAAATTCTGGATTTGGATTTACAACATTTTTAAATGTTTCATCTTC

AAATGCATGCTCTTCAACCTCAATAACGTTATATCCTGATTCACGTAATATTTTTGGG

GTAAATTTAGTTCCTGTTCCATTAACTGCGCTAAAAATAATTTTTAAATCTTTTTTAG

CTTCTTGCTCTTTTTTGTACGTCTCT

(see e.g., the world wide web at (www)addgene.org/52961/).

It is also contemplated within the scope of the disclosure that the region of sequence overlap for the Gibson reaction may be at least 30 nucleotides in length.

In a second step, the intermediate nucleic acid sequence may be linearized by removing the unique linker, and a ligation reaction may then occur between the linearized intermediate nucleic acid sequence and a linker block having the structure, in 5' to 3' order: a first gRNA scaffold, a unique linker sequence, and a mouse U6 promoter.

An exemplary linker block may contain a first gRNA scaffold and mouse U6 promotor (shown in bold)(SEQ ID NO: 21):

GTTTAAGAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATC

AACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTCGAGTACTAGGATCCATTA

GGCGGCCGCGTCGACAAGCTTTCTAGAGAATTCGATCCGACGCGCCATCTCTAGG

CCCGCGCCGGCCCCCTCGCACGGACTTGTGGGAGAAGCTCGGCTACTCCCCTG

CCCCGGTTAATTTGCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCG

ATAAAAGACAGATAATCTGTTCTTTTTAATACTAGCTACATTTTACATGATAGG

CTTGGATTTCTATAACTTCGTATAGCATACATTATACGAAGTTATAAACAGCAC

AAAAGGAAACTCACCCTAACTGTAAAGTAATTGTGTGTTTTGAGACTATAAGTA

TCCCTTGGAGAACCACCTTGTTG

A complete exemplary linker sequence including leading and trailing sequences may contain the following sequence (SEQ ID NO: 22):

TATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAgaattaatttgactg taaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatat gcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgCCTCCCGCTCCTGGAGCGGG7^"7T/44 GAGCTAAGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTC GGT^"GC7T7TT7TCTCGAGTACTAGGATCCATTAGGCGGCCGCGTCGACAAGCTTTCTAGAGAATTCgatccgacgcgcc atctctaggcccgcgccggccccctcgcacggacttgtgggagaagctcggctactcccctgccccggttaatttgcatataatatttcctagtaacta tagaggcttaatgtgcgataaaagacagataatctgttctttttaatactagctacattttacatgataggcttggatttctataacttcgtatagcata cattatacgaagttataaacagcacaaaaggaaactcaccctaactgtaaagtaattgtgtgttttgagactataagtatcccttggagaaccacct tgttgGATATTCACCATTATAGGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGA AAAAGTGGCACCGAGTCGGTGCI 1 1 1 1 / GAATTCTAGACTTGATGCTAACTAGGTCTTGAAAGGAGTGGGAATTG GCTCCGGTGCCCGTCAGT

(The human U6 promoter is shown in lowercase, mouse U6 promoter is shown in bold lowercase, gRNAl is shown in uppercase bold, gRNA2 is shown in uppercase bold italic, and the first and second scaffold sequences, respectively, are shown in uppercase italic).

Once the ligation reaction between the linearized intermediate nucleic acid sequence and a linker block is complete, a pgRNA library vector having a nucleic acid sequence including, in 5' to 3' order, a human U6 promoter, a first gRNA, a first gRNA scaffold, a unique linker, a mouse U6 promoter, a second gRNA, and a second gRNA scaffold is constructed (see e.g., FIG. 3E, lower panel).

Decoding the pgRNA libraries

The pgRNA libraries may be decoded by amplifying the pgRNA region from the plasmid or genomic DNA samples with the following exemplary primers:

pgRNA Lib F (SEQ ID NO: 23):

AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC TTTGTGGAAAGGACGAAACACCG

pgRNA Lib Rl (SEQ ID NO: 24):

TCTACTATTCTTTCCCCTGCACTGTACCCGGACTAGCCTTATTTTAACTTGCTATTTCT AGC TC T A A A AC

pgRNA_Lib_R2 (SEQ ID NO: 25):

CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTC CGATCTNNNNNNNNTCTACTATTCTTTCCCCTGCACTGTACC (N(8) is the specific index sequences)

The amplified pgRNA library may then be sequenced using any of a variety of high throughput sequencing techniques known in the art such as, for example, the Illumina high- throughput platform.

readl seq (SEQ ID NO: 26):

GAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT (for the IstgRNA)

read2_seq (SEQ ID NO: 27):

TGCACTGTACCCGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC (for the 2stgRNA)

index seq (SEQ ID NO: 28):

GCTAGTCCGGGTACAGTGCAGGGGAAAGAATAGTAGA

In a pilot scale pgRNA CRISPR screen using the above pgRNA library vector, a 7.5k pgRNA library was used to delete regulatory cis-elements in a human breast cancer line T47D. The sequencing data of the vector library and cell library by our new paired-end sequencing method demonstrated that that library quality was very high and that there was minimal recombination between the two gRNAs. As shown in FIG. 3G, the method of vector construction depicted in FIG. 3E reduces frequencies of recombination/swapping of pgRNAs during library construction.

Example 4: Design and Construction of the SLGI pgRNA CRISPR Library

A pgRNA CRISPR library was synthesized in an "a x b" design to explore all genetic interactions between anchors (i.e., part "a") and partners (i.e., part "b") using an improved oligo design with the following general structure: "gRNAl + unique linker + gRNA2". Part "a" may include four TSGs including Phosphatase and Tensin Homolog (PTEN), Neurofibromin 1 (NFl), RB Transcriptional Corepressor 1 (RBI), C-Src Tyrosine Kinase (CSK), as well as one control anchor, AAVS1, that has no function in the genome. Part "b" may include 121 genes that encode kinases and are targets of approved drugs according to annotations in the OASIS database (see e.g., reference 30), as well as AAVS1 as a control.

In an exemplary embodiment, the screen was carried out in a breast cancer cell line, T47D, in which no mutations are detected in any of the four TSG anchors. Between each anchor- partner pair, 21 pgRNA pairs may be designed. Advantageously, this number of pgRNA pairs conveniently fit in one 15K Agilent oligo synthesis order (21 * (4+1) * (121+1) < 15K). Each gene has 7 unique CRISPR gRNAs designed from an efficiency model (see e.g., reference 31) and validated recent screens. 21 pgRNA pairs were then selected according to the selection matrix from all 49 possible pairwise gRNA combinations (FIG. 14). The 15K pgRNA vector library was then constructed from the faithfully amplified oligo pool using the two-step cloning described in detail above. The lentivirus was packaged from the vector library and the four cell lines was infected at low MOI (-0.3) with 500-fold coverage to build the cell libraries with biological replicates.

Quality control was assessed for both plasmid and cell libraries by paired-end pgRNA sequencing to ensure the coverage and evenness of all designed pgRNAs and to check for swapping/recombination events (FIG. 15). The frequency of such swapping/recombination events were addressed by sequencing the library samples deeper to ensure sufficient coverage of the library after the swapped products have been eliminated. Example 5 : SLGI pgRNA CRISPR Screen

To screen for SLGI pairs that play key roles in cell growth, library cells were cultured at over 500-fold coverage of the library size for 11-12 population doubling times (~3 weeks for T47D cells) and the genomic DNA was harvested on day 0 and the end time point. After amplifying the pgRNA from all the samples, deep-sequencing libraries were prepared to submit for paired-end sequencing to decode the pgRNA information. The functional positive control SLGI pairs were confirmed in the screen, indicating that the screen works well (FIG. 16).

To call SLGI genes anchored on each TSG, the method based on regression residual was used, which is similar to the approach used in shRNA screens (see e.g., reference 9). The phenotype for each CRISPR gRNA in either the single (e.g., targeting gene X as a partner to AAVS1) or double (e.g., targeting gene X as a partner to TSG) KO was quantified as the fold change in gRNA abundance between selection and the day 0 control. For most of gRNAs, a linear relationship between the phenotype of the single and double KO is expected. Each gRNA on the partner and paired with a TSG gRNA may be ranked by the p-value (fold-change determines rank directions) of its deviations from the linear fit between double KO and single KO phenotype (FIG. 4; FIG. 17A-FIG. 17D). The top ranked SLGI pairs include RB1 MAPK8, RB1 JAK3, PTEN CDK12, PTEN AKT3, NF1 TYR03, NF1 EPHA5, CSK NTRK3 and CSK AR. Another method may adopt the BLISS independence model (see e.g., reference 32, incorporated herein by reference).

The techniques herein provide a robust pgRNA CRISPR screening technique, as well as a data analysis pipeline for SLGI identification.

The pgRNA CRISPR screening techniques described herein have the potential to create segmental genomic deletions in the situation where two gRNAs target a pair of genes that are in close proximity to one another. To avoid this confounding issue, all gene pairs that are within 1 mega base pair of one another in the library design may generally be excluded. An alternative strategy to study genetic interactions between proximal gene pairs is to use a CRISPR

interference screening technology that avoids genome cutting.

Another potential issue of an "a x b" paired design is that paired-end sequencing of the pgRNA may underestimate pgRNA swapping frequency from the sequencing preparation PCR step. However, as discussed above, use of an exo-polymerase may reduce the swapping rate by about 25% and top pgRNA hits can still be reliably identified. Even at swapping rate of about 50%, top pgRNA hits may still be identified because a particular swapped pair will only happen at a very low frequency, which is unlikely to overwhelm the frequency of the correct pgRNA pair.

Another potential issue may arise in the circumstance in which copy number alteration (CNA) confounds gene essentiality in a CRISPR screen (see e.g., reference 34). CNA may be addressed by the techniques here by assessing CNA profiles of screened cell lines in view of databases such as, for example, CCLE35 and GDSC databases (see e.g., reference 36), which may be used to reduce or eliminate the impact of CNA in determined pgRNA essentiality scores.

Example 6: Optimized sgRNA Design and Gene Calling for Genome-wide CRISPR Screens

Recent studies of CRISPR guide efficiency have analyzed the growth effects of different sgRNAs targeting genes that are essential for cell growth, and identified DNA sequence features that contribute to sgRNA efficiency in CRISPR-based screens (see e.g., reference 31).

Leveraging the information from multiple sgRNA library designs (see e.g., references 7, 21, and 22), the techniques herein provide a new sequence model for predicting sgRNA efficiency in CRISPR/Cas9 KO experiments. This model confirms known features and suggests new features that include, but are not limited to, a preference for cytosine at the cleavage site (FIG. 5 A). The model was experimentally validated for sgRNA-mediated mutation rate and gene KO efficiency (FIG. 5B) in that it achieved significant results under both positive and negative selection conditions, and clearly outperformed existing models (such as, e.g., those described in reference 37).

The ability to use CRISPR screen technology to identify CRISPR screen hits from sequencing data, a statistical algorithm, MAGeCK, Model-based Analysis of Genome-wide CRISPR/Cas9 KO has been developed (see e.g., reference 33). The MAGeCK algorithm was expanded via an updated algorithm, MAGeCK -VISPR, which provides a comprehensive quality control (QC), analysis, and visualization workflow for CRISPR screen analysis (see e.g., reference 38). Given the design matrix annotating the different screen conditions (FIG. 5C), MAGeCK first uses the sequence model to estimate sgRNA efficiency. It then iteratively updates each sgRNA efficiency based on whether the sgRNA behavior follows the selection of the gene across conditions (see, e.g., the E step in FIG. 5D), and uses the updated sgRNA efficiency to estimate the level of gene selection in different samples (see, e.g., the M step in FIG. 5D). Example 7: Novel Algorithm to Predict SLGI Pairs

The present disclosure provides a new algorithm for SLGI prediction. About 5,000 experimentally validated SLGI pairs in yeast (see e.g., reference 1) were assembled and their corresponding orthologous human genes were identified. The patterns of gene mutation, expression in TCGA, and protein-protein-interactions (PPI) of these orthologous genes were then examined. Using these yeast-to-human SLGI pairs as positive controls and 5,000 randomly selected gene pairs as negative controls, a feature selection and regression model was constructed to predict whether a pair of human genes will have SLGI. In this model, the response variable is whether the pair has SLGI, whereas the independent variables include expression, mutation, and CNV features of the two interacting genes in TCGA molecular profiles and PPI.

In TCGA breast, prostate, lung, and colon cancer data that was examined, the regression model consistently found the following factors to show statistical significance in predicting SLGI:

1) better overall positive expression correlation in the tumor samples;

2) more PPI;

3) better positive fold change (tumor-to-normal) correlation than random pairs;

4) when one SLGI gene is frequently deleted in cancer, the expression of the other often significantly increases; and

5) when one SLGI gene shows down-expression in tumor over normal, the expression correlations of the pairs tend to be negative.

Using the yeast SLGI and TCGA data for training and one of the very few available mammalian high throughput experimental genetic interaction (GI) screens for testing (see e.g., reference 11), it was found that the new algorithm disclosed herein provides a statistically significant separation between the genetically interacting (GI) pairs and non-GI pairs (FIG. 6).

The techniques herein provide that the new SLGI prediction algorithm may be refined/improved in a variety of ways. For example, more independent variables (features) for testing and selection may be included in our regression model. Such independent variables may include, but are not limited to, correlations of expression and mutations (including CNA) in different TCGA cancer types, frequency of mutations or differential expression in TCGA, as well as the association of a gene's expression or mutation with patient prognosis. This may allow SLGI pairs that have robust relationship to be identified across most TCGA cancer types, as well as those unique to certain cancer types. The RABIT method may be to select those independent variables (features) that are predictive of SLGI (see e.g., reference 39). RABIT utilizes the efficient Frisch-Waugh-Lovell theorem to correct confounding effects in linear models for fast stepwise feature selection.

As another example, efficiency of the prediction algorithm may be increased by using more SLGI data, which may include pgRNA CRISPR SLGI screening data and "1 x n" design CRISPR SLGI screening data. Additionally, efficiency of the prediction algorithm may be increased by adding known SLGI pairs in yeast and C. elegans that have orthologous genes in human, literature-reported SLGI individual genes in mammalian genomes, as well as the previous shRNA screens for SLGI (e.g., SynLethDB40). The regression model may be trained on each known SLGI dataset separately, evaluated for its performance using 10-fold cross validation (CV), and each dataset may be assigned a specific weight based on the CV R2 metric. Then, all the known SLGI datasets may be combined into one feature selection and regression model, with weights assigned to each dataset proportional to its cross-validation performance (FIG. 7). Preliminary testing conducted by adding new features (e.g., PPI) or data (e.g., combining yeast SLGI pairs with human colon cancer shRNA screen), the new algorithm may improve the area under the curve (AUC) on the receiver operating characteristic (ROC) curve by > 0.1 to final AUC > 0.7.

The above described SLGI algorithm may predict a likelihood of SLGI between every pair of human genes in each cancer type. However, the specific expression and mutation profiles in a particular patient tumor or cancer cell line dictate a tumor- or cell-line specific prediction of SLGI. For each cell line or tumor sample of a specific cancer type, the molecular profiles may be examined and an activity score for each gene may be computed based on its molecular profiles in the tumor. Low activity scores reflect copy number deletion, nonsense/frameshift mutations, or lower expression level, while high activity scores represent copy number amplification, known gain-of-function mutations, or higher expression level. Then, for each SLGI, its predicted likelihood may be re-weighted by the minimum activity score of the two partner genes. The accuracy of this tumor-specific SLGI prediction may be evaluated by cross validation as described below.

The present computational algorithm provides significant advantages over prior art SLGI prediction algorithms (see e.g., reference 20) in a number of ways. First, the regression model may consider many more public data and features and use feature selection to select those that are associated with SLGI. Second, weights may be given to the response variable in the different training data based on the confidence and strength of the observed SLGI. Finally, instead of using a number of Wilcoxon rank sum tests to filter gene pairs which could falsely remove promising pairs on one specific feature (as described in reference 20), the present multiple regression model automatically assigns feature weights, removes redundant features, and assigns a quantitative confidence for each prediction.

Example 8: Cross-validation to Systematically Evaluate New Algorithm Performance

Data has been collected for yeast-to-human SLGI pairs, as well as human SLGI pairs identified in previous literature studies, shRNA screens, and CRISPR screens on isogenic cell lines. The above-described TSG anchored SLGI genome-wide screening data may provide one additional high quality dataset with which to further evaluate the new SLGI prediction algorithm. The performance of the new algorithm may be systematically validated through a three-fold cross-validation (CV) procedure. The algorithm may initially be trained based on two-third SLGI pairs and used to predict the likelihood of SLGIs for the one-third held-out data and to then evaluate the prediction accuracy. In addition, CV may also be done by leaving one data set (e.g., an isogenic cell line screen for one TSG) out to validate the models trained on all other data sets. Based on the CV R2 metric, the SLGI prediction performance may be further compared between the new algorithm disclosed herein and previous algorithms (see e.g., reference 20 and 16).

In addition to evaluating SLGI prediction performance, the CV R2 metric may also be used to estimate the effect of down-sampling pgRNA pair number. Using the above-described pgRNA screening data, the number of pgRNAs for each gene pairs may be down-sampled and used to compute the CV R2 metric. If a significant deterioration of CV R2 is observed at certain pgRNA number, a higher number of pgRNA may be used in a design for large scale validation.

The new computational algorithm described above may be further refined to predict SLGI pairs in the human genome by integrating existing SLGI knowledge, high throughput SLGI identification data from previous literature and CRISPR screens, as well as TCGA data. The above described techniques may also be used for high throughput experimental validation of predicted SLGI pairs, without anchoring on one TSG in isogenic cell lines. It should be noted that many cancer cell lines harbor mutations and CNVs already, and thus SLGI pairs with one gene already mutated in these cell lines might display an unexpected behavior. For example, PTEN has a heterozygous deletion in the LNCaP cell line, so genes with SLGI with PTEN might not show a strong difference in phenotype between single KO and double KO (targeting PTEN and its SLGI partners) screens. Similarly, unique SLGI behavior may be observed between LNCaP (prostate) and ZR-75-1 (breast), not due to their tissue of origin, but due to the unique mutations intrinsic to these two cell lines. Thus, when using the cell line screening data to either train or validate the new computational algorithm, it is necessary to consider the confounding effects of cell line specific genetic backgrounds. Since the somatic mutation and copy number information for most COSMIC cell lines are measured (see e.g., reference 36), it may be necessary to remove genes mutated or deleted in a cell line in the process of computational method training and validation.

Example 9: Expanded SLGI Knowledge Base

As described above, initial screens only tested the potential SLGI between 4 tumor suppressor genes (TSGs) and about 700 druggable genes. Many other TSGs are frequently lost as a result of mutation/deletion/inactivation in many cancers, and it has not been possible so far to restore their functions in the clinic. Therefore, it is critical to identify the SLGI partners of TSGs, which may enable therapies to treat cancers with TSG loss. The novel TSG SLGI partners identified without available inhibitors may be important new targets for drug development.

Furthermore, different cancers and different tumors of the same cancer type likely have distinct transcriptome and mutation profiles, which may lead to cancer- or tumor-specific SLGI pairs. The above described SLGI-prediction algorithm has the advantage of being able to account for these differences by integrating cancer-specific and cell-specific genetic alteration and gene expression, among other factors, into the prediction of new SLGI pairs.

Example 10: Large Scale SLGI Screening Across Five Cancer Types

The techniques described herein may generate pan-cancer, cancer-specific as well as cell line-specific SLGI across all the human genome across all TCGA cancer types. To

systematically evaluate the predictions, especially the novel ones, a CRISPR SLGI screening strategy targeting specific gene pairs predicted by our algorithm may be used in about 20 cancer cells across about 5 cancer types. The pgRNA screening library may include candidate pan- cancer, cancer-specific, as well as cell-specific SLGI pairs involving -50 TSGs, consisting of -4K pairs across different scores of prediction confidence. More pgRNA pairs may be designed to target the more confident predictions, and the specific number of pgRNA pairs as well as the number of pgRNAs / pair in the CRISPR library design may be based on the power analysis described above. pgRNA CRISPR library construction and screening may be done as described above. The analysis to call SLGI depends on the number of predicted SLGI partners tested in the pgRNA CRISPR screen: a regression residual approach may be used for TSGs with many tested partners, while a BLISS independence model may be used for TSGs with fewer tested partners.

The results of these screens may significantly expand our knowledge of SLGI in different cancers and reveal potential novel therapy targets in cancers with non-targetable loss-of-function mutations. Additionally, examining the SLGI hits within the predicted pan-cancer SLGI, cell- specific SLGI, and non-SLGI may further evaluate the sensitivity and specificity of the new prediction algorithm, and assess its general applicability in target identification of cancer.

Furthermore, the data generated herein may also serve as new training data to refine our algorithm.

Example 11 : Characterizing the Mechanisms of Pan-Cancer and Cell-Specific SLGIs

Based on the above-validated SLGIs, two SLGI pairs each in the pan-cancer or cell- specific categories (FIG. 8A) may be selected and assessed for their respective mechanisms. Priority for selection may be given to novel SLGI pairs with frequent TSG loss in cancers and partners with available inhibitors. For the selected SLGI pairs with TSG "A" and druggable gene "B," small molecule inhibitors against B may be tested to determine if they have stronger killing in the cells harboring inactivating mutations in TSG "A." In addition, RNA-seq may be performed on unperturbed, gene "A" single KO, gene "B" single KO, or double "A+B" KO in two cell lines of different cancer type, respectively. Analysis of the RNA-seq may identify the transcriptome programs uniquely altered in the double KO condition, which might underlie the SLGI in different cancers or cell lines. Some pathways essential for cell survival or proliferation may remain unaffected or even activated with single gene KO, but be inactivated or inhibited with double KO in the SLGI pair. This may be assessed by validation assays. For example, in the case of a specific pan-cancer SLGI pair with TSG A and partner B, literature and pathway analysis may be conducted to examine whether the two genes share downstream pathways. If so, such pathway activity may be tested to determine if it is significantly altered only when both A and B are deficient and whether modulating its activity can influence the synthetic lethality (FIG. 8B). From the RNA-seq profiles above, perturbed pathways may be assessed by enrichment algorithms such as GSEA (see e.g., reference 41), GO analysis (see e.g., reference 42), and GREAT (see e.g., reference 43). Usually many of the downstream genes will also be SLGI hits, albeit weaker, which may be confirmed either from predictions or from available CRISPR screening results. NEST (see e.g., reference 44) analysis may be applied to determine whether SLGI prediction or differentially expressed genes are enriched for PPI members. The identified pathways serve as putative mediator(s) of SLGI, and may be assessed by genetic or

pharmacological modulations.

For a cancer type-specific SLGI pair with TSG "C" and partner "D" genes, there are two general scenarios: the "CD" downstream pathway is differentially expressed (FIG. 8C left), or they are similarly expressed but differentially required (FIG. 8C right). Published CRISPR screens have shown that if members of a protein complex involving gene "D" are all up- regulated in expression, "D" can be more essential without being differentially expressed (see e.g., reference 44). RNA-seq profiles may pinpoint the underlying scenario. For "CD" downstream pathways differentially required, a NEST analysis may be applied to the expression data to examine whether the differential expression of the PPI partners of "C" or "D" cause them to be differentially required. For "CD" downstream pathways differentially expressed, the expression profile and transcriptional regulatory network may be used to identify their upstream regulators that are differentially expressed in different cancers. These techniques may utilize any of a variety of algorithms (e.g., MACS 45, Cistrome AP 46, RABIT 39, MARGE 47, and the like) and databases (e.g., Cistrome DB 48) for transcription regulation. Identified transcriptional regulators that underlie the differential pathway may be verified by using genetic perturbation to verify their role in mediating the cancer type-specific SLGI relationship.

It should be noted that double KO of SLGI genes may lead to dramatic cell death and/or senescence by definition. Consequently, gene expression profiling of such double KO cells may become technically infeasible with limited cell number. To overcome this caveat, alternative approaches may be used to perturb the candidate genes for expression profiling such as, for example, by (inducible) RNAi or small molecule inhibition. However, it is not uncommon for RNAi and small molecule inhibitors to have pleiotropic or off-target effects, so it is possible that different phenotypes may be observed between functional validations using shRNA and/or small molecule inhibitors versus pgRNA-mediated double KO. To ensure robustness of the validation, multiple small molecule inhibitors or multiple shRNAs may be tested against the candidate genes. Additionally, exome and cistrome genotypes in these cancer cell lines may be the confounding factors that affect the interpretation of the SLGI screening data, so choosing cancer cell lines that have exome sequencing and copy number variation data available from COSMIC and CCLE to ensure that this information could be taken into consideration.

Example 1 1 : A paired-guide (pgRNA) CRISPR Library for Functional Enhancer Screen

The techniques herein also provide that a paired-guide CRISPR library may be used to conduct functional enhancer screen(s). As shown in FIG. 9A, the rationale of the strategy is that two gRNAs may be introduced into a single cell, and if the two targeting loci are close to each other, then the fragment in-between has a high probability of being deleted, rather than having two indels mutation at each of the two loci separately. Because the deletion could affect larger regions than small indel mutations, the techniques herein provide that a small number of pgRNAs may be used to cover much larger regions of the genome than sgRNA libraries.

Furthermore, since the deletion could completely knock out the putative functional motifs of an enhancer, the efficiency is also higher.

For an enhancer screen experiment, a small pgRNA library containing 7500 pairs of guide RNAs was designed for use in screening in an ER+ breast cancer cell line: T47D. This line had previously been used to conduct a genome-wide CRISPR screens. In an exemplary embodiment, the distance range between the two gRNAs was between 150-300 bp.

This library was designed to target three groups of predicted cis-elements: 1) Enhancers and promoters of positively-selected genes: PTEN, TSC1, RBI, CSK (tilling arrays); 2) Enhancers and promoters of negatively-selected genes: ESR1, MYC, GATA3, FOXA1; and 3) A short list of CTCF and FOXA1 binding sites from the sgRNA CRISPR library. An overview of the screening procedure is shown in FIG. 9B, in which the cell libraries were cultured for 30 days under three conditions: full medium, white medium and white medium + Estrogen (E2) before harvested for genomic DNA and sequencing of the pgRNAs together with the Day 0 cell library sample as control. Negative controls used in the enhancer screen included double cuts on AAVS1, whereas positive controls used in the enhancer screen included double cuts on an essential gene + AAVS1.

As shown in FIG. 10, CSK is an important positively-selected gene in T47D and MCF7 cell lines under hormone-depleted growth condition (also shown in FIG.2C). Knockout of the putative CSK enhancer with ER binding and DNase-I/H3K27ac mark totally abolished CSK expression upon estrogen stimulus (FIG. 10 right panel). Therefore, CSK enhancer loss reconstructs the CSK -knockout phenotype under estrogen-depleted growth condition.

FIG. 11 shows an exemplary tilling design to target the CSK enhancer, in which more than 1,300 pgRNAs were designed in a tilling format to cover the CSK enhancer region in which each pgRNA flanks 150-300 bp locus to search for novel and unknown CSK enhancers.

The CSK enhancer tilling design shown in FIG. 1 1 was analyzed by a modified

MAGeCK algorithm with conversion of pgRNAs into consecutive bins of DNA locus, result in a representative p-value plot of each bin to show a potential functional enhancer, as shown in FIG. 12.

As shown in FIG. 13, the functional enhancer screen successfully identified known CSK enhancers, as well as potentially novel enhancer elements. As the positive selection p-value plot shows, the three peaks represent one functionally validated CSK enhancer co-localized with DNase-I/H3K27ac mark and ESR1 -binding peak (FIG. 10) and two previously unknown enhancers with only H3K27ac marks.

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INCORPORATION BY REFERENCE

All documents cited or referenced herein and all documents cited or referenced in the herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated by reference, and may be employed in the practice of the disclosure.

EQUIVALENTS

It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the disclosure. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS We claim:

1. A paired-guide ribonucleic acid (pgRNA) vector, comprising:

a first guide RNA (gRNA) cassette;

a second gRNA cassette; and

a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) expression cassette;

wherein the second gRNA cassette is positioned between the first gRNA cassette and the Cas9 expression cassette.

2. The pgRNA vector of claim 1, wherein the first gRNA cassette includes a first nucleic acid sequence including, in 5' to 3' order, a first gRNA promoter, a first gRNA, and a first gRNA scaffold, and the second gRNA cassette includes a second nucleic acid sequence including, in 5' to 3' order, a second gRNA promoter, a second gRNA, and a second gRNA scaffold.

3. The pgRNA vector of claim 2, wherein the first gRNA promoter is selected from the group consisting of a mouse U6 promoter, a human U6 promoter, a modified bovine U6 promoter, a mouse HI promoter, a human HI promoter, a mouse 7SK promoter, and a human 7SK promoter, and a modified bovine 7SK promoter.

4. The pgRNA vector of claim 2, wherein the second gRNA promoter is selected from the group consisting of a mouse U6 promoter, a human U6 promoter, a modified bovine U6 promoter, a mouse HI promoter, a human HI promoter, a mouse 7SK promoter, and a human 7SK promoter, and a modified bovine 7SK promoter.

5. The pgRNA vector of claim 2, wherein the second gRNA promoter is different than the first gRNA promoter.

6. The pgRNA vector of claim 2, wherein the first gRNA and the second gRNA are each between about 17 and 27 nucleotides in length.

7. The pgRNA vector of claim 2, wherein the first gRNA and the second gRNA are each about 19 nucleotides in length.

8. The pgRNA vector of claim 1, wherein the pgRNA vector is constructed by using an intermediate pgRNA nucleic acid, comprising:

a first guide RNA (gRNA);

a unique linker; and

a second gRNA;

wherein the unique linker is positioned between the first gRNA and the second gRNA .

9. The pgRNA vector of claim 8, wherein the unique linker is about 16 nucleotides in length.

10. The pgRNA vector of claim 1, wherein the Cas9 cassette includes a promoter, a Cas9 coding sequence, and a P2A sequence.

11. A method of making a paired-guide RNA (pgRNA) library vector, comprising:

obtaining a first nucleic acid sequence including, in 5' to 3' order, a first guide RNA (gRNA) cassette promoter, a vector linker, and a second gRNA cassette scaffold;

removing the vector linker to create a double strand break (DSB) between a 3' end of the first gRNA cassette promoter and a 5' end of the second gRNA cassette scaffold;

inserting into the DSB a second nucleic acid sequence including, in 5' to 3' order, a first guide RNA (gRNA) sequence, a unique linker, and a second gRNA sequence to create an intermediate nucleic acid sequence;

removing the unique linker to create a DSB in the intermediate nucleic acid sequence between a 3' end of the first gRNA sequence and a 5' end of the second gRNA sequence;

inserting into the DSB in the intermediate nucleic acid sequence a third nucleic acid sequence including, in 5' to 3' order, a first gRNA cassette scaffold, a spacer, and a second guide RNA (gRNA) cassette promoter, thereby creating the pgRNA vector.

12. The method of claim 11, wherein the first gRNA cassette promoter is selected from the group consisting of a mouse U6 promoter and a human U6 promoter.

13. The method of claim 11, wherein the second gRNA cassette promoter is selected from the group consisting of a mouse U6 promoter and a human U6 promoter.

14. The method of claim 13, wherein the second gRNA cassette promoter is different than the first gRNA cassette promoter.

15. The method of claim 11, wherein the first gRNA sequence and the second gRNA sequence are each between about 17 and 27 nucleotides in length.

16. The method of claim 15, wherein the first gRNA sequence and the second gRNA sequence are each about 19 nucleotides in length.

17. The method of claim 11, wherein the unique linker is between about 12 and 24 nucleotides in length.

18. The method of claim 11, wherein the unique linker is about 16 nucleotides in length.

19. The method of claim 11, wherein the first nucleic acid sequence further includes a Cas9 cassette.

20. The method of claim 19, wherein the Cas9 cassette includes a promoter, a Cas9 coding sequence, and a P2A sequence.

21. A paired-guide RNA (pgRNA)/Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR) library, comprising: a plurality of pgRNA sequence pairs capable of targeting a plurality of target sequence pairs in a target genome via a CRISPR/Cas9 system to knockout function of a first target sequence and a second target sequence in the target sequence pair, wherein pgRNA vector is constructed by using an intermediate pgRNA nucleic acid, that includes a first guide RNA (gRNA); a unique linker; and a second gRNA; wherein the unique linker is positioned between the first gRNA and the second gRNA.

22. The pgRNA/CRISPR library of claim 21, wherein each of the plurality of pgRNA sequence pairs includes a first guide RNA (gRNA) cassette and a second gRNA cassette.

23. The pgRNA/CRISPR library of claim 22, wherein the first gRNA cassette includes a first nucleic acid sequence including, in 5' to 3' order, a first gRNA promoter, a first gRNA sequence, and a first gRNA scaffold, and the second gRNA cassette includes a second nucleic acid sequence including, in 5' to 3' order, a second gRNA promoter, a second gRNA sequence, and a second gRNA scaffold.

24. The pgRNA/CRISPR library of claim 23, wherein the first gRNA promoter is selected from the group consisting of a mouse U6 promoter and a human U6 promoter.

25. The pgRNA/CRISPR library of claim 23, wherein the second gRNA promoter is selected from the group consisting of a mouse U6 promoter and a human U6 promoter.

26. The pgRNA/CRISPR library of claim 25, wherein the second gRNA promoter is different than the first gRNA promoter.

27. The pgRNA/CRISPR library of claim 23, wherein the first gRNA sequence and the second gRNA sequence are each between about 17 and 27 nucleotides in length.

28. The pgRNA/CRISPR library of claim 23, wherein the first gRNA sequence and the second gRNA sequence are each about 19 nucleotides in length.

29. The pgRNA/CRISPR library of claim 21, wherein the unique linker is between about 12 and 24 nucleotides in length.

30. The pgRNA/CRISPR library of claim 21, wherein the unique linker is about 16 nucleotides in length.

31. A method of identifying synthetic lethal genetic interactions (SLGIs) or enhancers within a genome, comprising:

contacting a population of cells with one or more pgRNA vectors of claim 1;

selecting successfully transduced cells;

culturing the population of cells for a plurality of population doubling times, wherein genomic DNA is harvested on a first day of culture and on a last day of culture;

deep sequencing the genomic DNA harvested on the first day of culture and on the last day of culture;

quantifying abundance of the pgRNAs at the first day of culture and the last day of culture;

analyzing an abundance fold change of the pgRNAs between the first day of culture and the last day of culture; and

identifying, based on the abundance fold change; a SLGI or enhancer.

32. The method of claim 31, wherein analyzing further includes a regression residual analysis.

33. The method of claim 31, wherein analyzing further includes a BLISS independence model analysis.

34. The method of claim 31, wherein the plurality of population doubling times is between about 8 and 16.

35. The method of claim 31, wherein the plurality of population doubling times is about 12.

36. A tangible, non-transitory, computer-readable media having software encoded thereon, the software, when executed by a processor on a particular device, operable to:

identify a plurality of gene pairs;

determine a response variable; analyze, by a feature selection and regression model, the plurality of gene pairs; and determine, based on the response variable and the analysis, that one or more gene pairs within the plurality of gene pairs interact genetically.

37. An intermediate paired-guide RNA (pgRNA) nucleic acid, comprising:

a first guide RNA (gRNA) cassette;

a unique linker; and

a second gRNA cassette;

wherein the unique linker is positioned between the first gRNA cassette and the second gRNA cassette.

38. The intermediate pgRNA nucleic acid of claim 37, wherein the first gRNA cassette includes a first nucleic acid sequence including, in 5' to 3' order, a first gRNA promoter, a first gRNA, and a first gRNA scaffold, and the second gRNA cassette includes a second nucleic acid sequence including, in 5' to 3' order, a second gRNA promoter, a second gRNA, and a second gRNA scaffold.

39. The intermediate pgRNA nucleic acid of claim 37, wherein the first gRNA promoter is selected from the group consisting of a mouse U6 promoter, a human U6 promoter, a modified bovine U6 promoter, a mouse HI promoter, a human HI promoter, a mouse 7SK promoter, and a human 7SK promoter, and a modified bovine 7SK promoter.

40. The intermediate pgRNA nucleic acid of claim 37, wherein the second gRNA promoter is selected from the group consisting of a mouse U6 promoter, a human U6 promoter, a modified bovine U6 promoter, a mouse HI promoter, a human HI promoter, a mouse 7SK promoter, and a human 7SK promoter, and a modified bovine 7SK promoter.

41. The intermediate pgRNA nucleic acid of claim 37, wherein the second gRNA promoter is different than the first gRNA promoter.

42. The intermediate pgRNA nucleic acid of claim 37, wherein the first gRNA and the second gRNA are each between about 17 and 27 nucleotides in length.

43. The intermediate pgRNA nucleic acid of claim 37, wherein the first gRNA and the second gRNA are each about 19 nucleotides in length.

44. The intermediate pgRNA nucleic acid of claim 37, wherein the unique linker is between about 10 and 30 nucleotides in length.

45. The intermediate pgRNA nucleic acid of claim 37, wherein the unique linker is about 16 nucleotides in length.

46. The intermediate pgRNA nucleic acid of claim 37, wherein the unique linker has a GC content less than or equal to 40%.