CN111979226B - Method capable of carrying out in-vitro off-target detection and sgRNA screening in batch - Google Patents
Method capable of carrying out in-vitro off-target detection and sgRNA screening in batch Download PDFInfo
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Abstract
A method capable of carrying out in-vitro off-target detection and sgRNA screening in batches relates to the technical field of genetic engineering. The invention aims to solve the problems that the existing sgRNA off-target detection method can only detect whether one sgRNA is off-target every time and how to screen the sgRNAs with high cutting efficiency and low off-target. The invention provides a high-throughput sgRNA off-target detection method, which is characterized in that in-vitro transcription is carried out in a sgRNA pool form, and the in-target or off-target condition of thousands of sgRNAs can be detected simultaneously; and judging the cutting efficiency according to the number of target reads, and further judging the height of the off-target effect according to the off-target sites and the number of the off-target reads. The invention can obtain a method for batch in-vitro off-target detection and sgRNA screening.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method capable of carrying out in-vitro off-target detection and sgRNA screening in batches.
Background
The CRISPR-Cas9 system is derived from acquired immune systems of bacteria and archaea, and becomes a powerful gene editing tool due to simple and efficient operation. Since the discovery that CRISPR/Cas9 achieves precise editing of DNA within cells in early 2013, blowout-type development has been presented in recent years. Compared with gene editing technologies such as ZFN, TALEN and the like, the method is simpler, more economical and easier to operate, is the most effective, cheap and easier gene editing method so far, and has extremely important application significance in gene editing (comprising gene knockout, knock-in, point mutation), gene therapy (sickle cell anemia HBB gene repair and the like), live cell imaging (such as Cas-FISH), functional gene screening (CRISPR/a positive and negative screening), target capture (CRISPR-capture) and the like. However, behind its widespread use, the CRISPR/Cas9 system has a fatal drawback of being prone to off-target, i.e., producing additional DNA cleavage or editing at sites other than the intended target. Off-target can cause unintended gene mutation and even cause carcinogenesis, limiting the clinical application of CRISPR gene editing.
Off-target, however, is a major drawback of the application of CRISPR/Cas9 systems for gene therapy and general applications. There are two main factors that contribute to the occurrence of off-target. The first is the fault tolerance of the sgRNA sequence, namely the sgRNA can be combined with a target point which is completely complementary and paired and cut, and can also be combined with other similar sequences on a genome to cut, and the fault tolerance can reach 8 bases or even more; second, sustained expression of Cas9 protein is susceptible to cleavage of non-target sites. Therefore, off-target is the key to influence clinical treatment effect and experimental result reliability in both clinical transformation of CRISPR gene therapy and routine experimental application. The key is how to screen the sgRNA with the highest cleavage efficiency and the lowest off-target reaction. On the other hand, for some monogenic genetic diseases, the position of a mutation site is fixed, the number of available sgrnas is limited, the off-target effect of the sgrnas needs to be determined in advance, and the sgrnas causing severe off-target are avoided as much as possible, which depend on off-target detection.
The detection techniques for off-target sites currently used in genome-wide are broadly divided into three categories: 1. software prediction + sequencing verification, for example, using Cas-OFF finder software to predict possible OFF-target of sgRNA, and further performing Sanger sequencing verification; 2. the off-target detection method based on the cell transfection technology comprises GUIDE-seq, BLESS, HTGTS, DISCOVER-seq and the like; 3. independent of cell technology, in vitro detection methods, including digomer-seq, SITE-seq and CIRCLE-seq. However, the above techniques all have certain limitations:
1) And (3) predicting by software: early OFF-target detection technologies consist of software prediction and sequencing (such as Sanger sequencing, NGS sequencing, whole exome sequencing and the like), and the technologies utilize Cas-OFF finder and other OFF-target prediction software to predict firstly to obtain possible OFF-target sites, and then perform PCR amplification and sequencing on the predicted OFF-target sites so as to determine whether the sites generate OFF-target mutation. The principle is to sequence on the basis of known off-target sites to determine which sites are edited. This type of technology has the disadvantages of low throughput and significant bias.
2) In vivo off-target detection: in vivo off-target detection methods based on cell transfection techniques lack operability for cell lines that are not easily transfected. Both GUIDE-seq dependent integration of the donor sequence dsODN into the genome and HTGTS dependent chromosomal translocation, both influenced by factors such as cell line DNA repair specificity and timeliness, target site specificity, cell cycle, etc., while off-target sites with less than 0.1% mutation frequency were missed because dsODN was not integrated or chromosomal translocation did not occur at the cleavage site. The DISCOVER-Seq detects the enrichment of a DSB repair factor MRE11 generated after the Cas enzyme is cut at a cutting site by using a ChIP-Seq method, thereby obtaining cutting site information. However, since the Cas enzymes do not cleave different sites at the same time, but have a chronological order, the differential detection result of MRE11 factor enrichment at the same time point does not truly reflect all cleavage sites. In addition, because a plurality of DSBs generated by non-Cas enzyme cleavage exist in the cells, the DSBs also become false positive sites during detection.
3) In-vitro off-target detection: compared with an in-vivo off-target detection method, the in-vitro off-target detection method can improve the detection repeatability, avoids the influence of cell transfection efficiency and cell repair on detection, can greatly improve the concentrations of sgRNA and Cas enzyme in an in-vitro experiment, and is favorable for detecting low-frequency mutation sites. At present, 3 methods for detecting off-target in vitro genome-wide, the earliest Digenome-seq detection scheme is that target DNA is cleaved by Cas enzyme exosomally, all free ends (whether DSB generated by Cas enzyme cleavage or not) are treated by adding linkers, and the on-target and off-target sites generated by Cas enzyme cleavage are analyzed by high-throughput genome-wide sequencing. However, the method needs a very high sequencing depth, has a large amount of random background signals generated by non-Cas enzyme cleavage, lacks sensitivity for comprehensively detecting all off-target sites including low-frequency sites, and is not suitable for large-scale sgRNA screening. And the subsequent SITE-seq detection scheme adopts high molecular weight genome DNA as a Cas enzyme cutting substrate, and carries out biotin labeling, enrichment, sequencing and comparison on the cut genome DNA to obtain comprehensive cutting SITE information. The method eliminates DSB generated by partial non-Cas enzyme cutting, and the required sequencing depth is obviously reduced compared with Digenome-seq, but the method has the defects that high molecular weight genome DNA can only come from fresh tissues and cells, the extraction is difficult, the integrity of the DNA is difficult to ensure, a large amount of DSB can be generated in the process of extracting the DNA, and finally the false positive rate of an analysis result is high. CIRCLE-seq detection protocol, which occurs contemporaneously with SITE-seq, utilizes circularized DNA as a cleavage substrate to remove background DSB. The method comprises the steps of connecting broken genome DNA with a special hairpin-like joint, then carrying out enzyme digestion on a stem-loop structure, then connecting the head and the tail of the DNA into a loop by using DNA ligase, then externally cutting the loop DNA by using a Cas enzyme, and forming a sequencing-capable library for sequencing comparison after the linearized DNA is connected with a sequencing joint.
Besides the disadvantages of the methods, another considerable problem exists, namely a single sgRNA detection mode, which can detect off-target of only 1 sgRNA each time, and has high cost, low flux and long experimental period, thus greatly limiting the application of CRISPR gene editing.
Disclosure of Invention
The invention aims to solve the problems that the existing sgRNA off-target detection method can only detect whether one sgRNA is off-target every time and how to screen the sgRNAs with high cutting efficiency and low off-target, and provides a method for carrying out in-vitro off-target detection and sgRNA screening in batches.
A method for batch in-vitro off-target detection and sgRNA screening is completed according to the following steps:
1. collecting 4X 10 6 ～8×10 6 Putting the individual cells into a centrifuge tube, centrifuging, removing the culture medium, washing once by PBS, centrifuging again, removing the supernatant, and extracting the cell genome DNA of the centrifuged solid phase substance;
2. breaking the genome DNA extracted in the step one into fragments of 300bp to 700bp, and then purifying by using DNA purification magnetic beads;
3. carrying out end repairing, tail adding and stem-loop structure joint 1 adding on the DNA purified in the step two, then adopting exonuclease treatment, and then using ddNTP treatment;
4. designing and synthesizing an sgRNA oligo library, and carrying out PCR amplification and in vitro transcription to obtain sgRNA pool;
5. carrying out in-vitro cutting on the DNA treated by the ddNTP in the step 3 by using Cas enzyme and sgRNA pool obtained in the step four;
6. carrying out end repairing, tail A adding and linear joint 2 adding on the DNA cut in vitro, wherein the 5' end of the linear joint 2 is modified by biotin;
7. resuspending streptavidin magnetic beads, adding 1 xBind and wash buffer, rotating, mixing and washing at room temperature, centrifuging, removing supernatant, repeating washing, centrifuging and removing supernatant for 3 times, resuspending the streptavidin magnetic beads by using 2 xBind and wash buffer, adding DNA cut in the fifth step, rotating and mixing at room temperature, placing on a magnetic frame, and removing supernatant to obtain DNA adsorbed by the streptavidin magnetic beads;
8. treating DNA adsorbed by streptavidin magnetic beads by using USER enzyme, cutting a stem-loop structure of the joint 1, performing recovery PCR amplification by using the DNA treated by the USER enzyme as a template, performing index PCR to obtain a library to be loaded, and performing library quality inspection and sequencing;
9. and after the library is off-line, performing bioinformatics analysis, and comparing each off-target site obtained by bioinformatics analysis with each sgRNA to obtain on-target reads and off-target sites corresponding to each sgRNA, the number of the on-target reads and off-target sites and corresponding reads information.
The invention has the beneficial effects that:
1. the invention relates to a method capable of carrying out in-vitro off-target detection and sgRNA screening in batches, and provides a high-flux sgRNA off-target detection method, which carries out in-vitro transcription in a sgRNA pool form and can simultaneously detect the in-target or off-target conditions of thousands of sgRNAs; the cutting efficiency can be judged according to the number of in-target reads, and the height of the off-target effect can be further judged according to the off-target sites and the number of off-target reads; compared with the traditional single sgRNA off-target detection mode with low flux, the flux is high.
2. The period is short: because most of the existing off-target detection methods are complex to operate, 8-12 sgrnas can be detected in each experiment at most, and if tens of thousands of sgrnas are required to be detected, multiple experiments are required, so that the experiment period is long. And the experimental batches are different, which results in unreliable results.
3. The cost is low: the cost of the previous single sgRNA off-target detection experiment is about 1 ten thousand yuan per case, and if off-target detection of a plurality of sgRNAs is carried out, the cost is very high; and by adopting the sgRNA pool off-target detection strategy, all the sgRNAs to be detected can be detected off-target at one time, only a single reaction is needed, and the experiment cost is greatly reduced.
4. The experimental result is reliable: since all sgrnas are performed under uniform experimental conditions, there is no difference between experimental batches, and the selection and comparison between sgrnas can be performed by a single experimental result.
5. The applicability is wide: one of the best examples is the CRISPR library screening currently in use, and the CRISPR libraries at present are mostly tens of thousands of sgrnas, each gene having a coverage of 4 sgrnas. However, since all sgrnas are not screened, some sgrnas have a large amount of off-targets, which greatly affects the accuracy of the experimental results. Therefore, the sgRNA with the highest cutting efficiency and the lowest off-target reaction of each gene is screened by using the sgRNA pool strategy, so that the experimental deviation caused by the off-target reaction can be greatly reduced.
6. Can be used for clinical treatment target screening: in recent years, CRISPR gene therapy is getting more popular, and diseases which can not be achieved by a plurality of traditional methods are expected to be treated by the CRISPR gene therapy. But firstly, target screening is needed, and the target with highest cutting efficiency and lowest off-target reaction is screened. For example, against HIV, HBV, HPV and the like, the target needs to cover the whole viral genome. If the traditional method is very time-consuming and labor-consuming, target spot screening can be completed at one time through the sgRNA pool off-target detection strategy.
7. Without species limitation: whether plants or animals can be detected; and can detect not only the off-target of SpCas9, but also the off-target of SaCas9, asCpf1 and LbCpf 1.
The invention can obtain a method for batch in-vitro off-target detection and sgRNA screening.
Drawings
Fig. 1 is a schematic flow chart of a method for batch in vitro off-target detection and sgRNA screening according to the present invention; a represents oligomeric synthesis, b represents PCR amplification, c represents in vitro transcription, d represents a sgRNA pool, e represents incubation with Cas9 or CPF1, f represents in vitro cleavage, and g represents AID-seq;
FIG. 2 is a diagram showing data for detecting the first position of the sgRNA of 74230 human genomes in the first example;
FIG. 3 is a diagram showing data for detecting a second site of the sgRNA of 74230 human genomes in the first example;
fig. 4 is a data diagram illustrating the detection of the third position of the sgRNA of 74230 human genomes in the first example.
Detailed Description
The first embodiment is as follows: the method for carrying out in-vitro off-target detection and sgRNA screening in batches comprises the following steps:
1. collecting 4X 10 6 ～8×10 6 Putting the individual cells into a centrifuge tube, centrifuging, removing a culture medium, washing once by PBS, centrifuging again, removing a supernatant, and extracting cell genome DNA of a centrifuged solid-phase substance;
2. breaking the genome DNA extracted in the step one into fragments of 300bp to 700bp, and then purifying by using DNA purification magnetic beads;
3. carrying out end repairing, tail adding and stem-loop structure joint 1 adding on the DNA purified in the step two, then adopting exonuclease treatment, and then using ddNTP for treatment;
4. designing and synthesizing an sgRNA oligo library, and carrying out PCR amplification and in vitro transcription to obtain sgRNA pool;
5. carrying out in-vitro cutting on the DNA treated by the ddNTP in the step 3 by using Cas enzyme and sgRNA pool obtained in the step four;
6. carrying out end repairing, tail A adding and linear joint 2 adding on the DNA cut in vitro, wherein the 5' end of the linear joint 2 is modified by biotin;
7. resuspending streptavidin magnetic beads, adding 1 xBind and wash buffer, rotating, uniformly mixing and washing at room temperature, centrifuging, removing supernatant, repeatedly washing, centrifuging and removing supernatant for 3 times, resuspending the streptavidin magnetic beads by using 2 xBind and wash buffer, adding DNA cut in the fifth step, rotating and uniformly mixing at room temperature, placing in a magnetic frame, and removing supernatant to obtain DNA adsorbed by the streptavidin magnetic beads;
8. treating DNA adsorbed by streptavidin magnetic beads by using USER enzyme, cutting a stem-loop structure of the joint 1, performing recovery PCR amplification by using the DNA treated by the USER enzyme as a template, performing index PCR to obtain a library to be loaded, and performing library quality inspection and sequencing;
9. and after the library is downloaded, bioinformatics analysis is carried out, each off-target site obtained by bioinformatics analysis is compared with each sgRNA, and in-target reads and off-target sites corresponding to each sgRNA, the number of the off-target sites and corresponding reads information are obtained.
The second embodiment is as follows: the present embodiment differs from the present embodiment in that: in the second step, DNA disruption is carried out by using an instrument Bioruptor, and parameters are as follows: 50 ng/. Mu.L of DNA; the volume is 100 mu L;15sON-90sOFF;6-8 times of circulation; the cleaved DNA was purified with DNA purification beads, and the elution volume was 37. Mu.L.
Other steps are the same as in the first embodiment.
The third concrete implementation mode: the first or second differences from the present embodiment are as follows: in the third step, a kit is adopted for DNA end repair, A tail addition and stem-loop structure joint 1 addition, wherein the specific reaction system and reaction program of the DNA end repair and the A tail addition are as follows:
the kit comprises: ABClonal rapid DNA library building kit
Reaction procedure: 30min at 20 ℃; obtaining a terminal repairing mixture at 65 ℃ for 30min;
the specific reaction system and reaction procedure of the stem-loop structure-added joint 1 are as follows:
reaction procedure: after 1h at 22 ℃ the column was purified with 1 XDNA purification beads, and the elution volume was 30. Mu.L.
The other steps are the same as those in the first or second embodiment.
The fourth concrete implementation mode: the difference between this embodiment and one of the first to third embodiments is: the reaction system and procedure of exonuclease digestion in step three are as follows:
reaction procedure: 2h at 37 ℃; after 10min at 75 ℃ the column was purified with 1 XDNA purification beads, eluting in a volume of 44. Mu.L.
The other steps are the same as those in the first to third embodiments.
The fifth concrete implementation mode: the difference between this embodiment and one of the first to fourth embodiments is: reaction system and procedure for ddNTP treatment in step three:
reaction procedures are as follows: at 75 ℃ for 30min; then using 1 x DNA purification magnetic beads purification, elution volume 25 u L, the Qubit measurement of DNA concentration >6.4 ng/. Mu.L.
The other steps are the same as those in the first to fourth embodiments.
The sixth specific implementation mode: the difference between this embodiment and one of the first to fifth embodiments is as follows: in the reaction system and procedure for in vitro cutting of the Cas enzyme in the fifth step, firstly, a sgRNA oligo library is designed and synthesized, and after PCR amplification, in vitro transcription is carried out to obtain active sgRNA pool, and the rest is as follows:
reaction procedure: combining for 10min at room temperature, then adding 50 ng-1000 ng of DNA treated in the third step, supplementing the volume to 50 mu L with enzyme-free water, incubating for 1h at 37 ℃, purifying with 50 mu LDNA purified magnetic beads, and eluting with 37 mu L.
The other steps are the same as those in the first to fifth embodiments.
The seventh embodiment: the difference between this embodiment and one of the first to sixth embodiments is: in the sixth step, an ABClonal rapid DNA library building kit is adopted for DNA end repair, A tail is added, and a linear joint 2 is added; the specific reaction system and reaction program for DNA end repair and A tail addition are as follows:
reaction procedure: reacting at 20 ℃ for 30min; reacting for 30min at 65 ℃ to obtain a terminal repairing mixture;
reaction procedure: after reaction at 22 ℃ for 1h, the column was purified using 1 XDNA purification beads, and the elution volume was 41. Mu.L.
The other steps are the same as those in the first to sixth embodiments.
The specific implementation mode is eight: the difference between this embodiment and one of the first to seventh embodiments is: adding the USER enzyme into the DNA obtained in the step five, reacting for 30min at 37 ℃, then placing the DNA in a magnetic frame for 2min, removing the supernatant, adding 10mM Tris-HCl, rotating, mixing uniformly at room temperature for 5min, placing the DNA in the magnetic frame for 2min, removing the supernatant, and washing for 2 times by using 10mM Tris-HCl in total; then 20. Mu.L of 10mM Tris-HCl was added for resuspension.
The other steps are the same as those in the first to seventh embodiments.
The specific implementation method nine: the difference between this embodiment and the first to eighth embodiments is: and the recovery PCR reaction system and the reaction program in the step eight are as follows:
reaction procedure: 45s at 98 ℃;12cycles of (98 ℃ 15s,61 ℃ 30s,72 ℃ 2 min), 72 ℃ 2min,4 ℃ hold, then placed in the magnetic frame for 2min, the supernatant transferred to a new Ep tube, 1. Mu.L-10. Mu.L of the supernatant diluted 50 times with water.
The other steps are the same as those in the first to eighth embodiments.
The detailed implementation mode is ten: the difference between this embodiment and one of the first to ninth embodiments is as follows: the reaction system and procedure for indexing the PCR in step eight are as follows:
reaction procedure: 45s at 98 ℃;12cycles of (98 ℃ 15s,60 ℃ 30s,72 ℃ 2 min), 72 ℃ 2min,4 ℃ hold, 0.7 XDNA purification magnetic bead purification, elution volume of 50. Mu.L, 0.6X-0.2 XDNA purification magnetic bead double-end screening, elution volume of 20. Mu.L, get 200-600bp DNA library.
The other steps are the same as those in the first to ninth embodiments.
The following examples were used to demonstrate the beneficial effects of the present invention:
the first embodiment is as follows: a method for carrying out in-vitro off-target detection and sgRNA screening in batches comprises the following steps:
1. collecting 4X 10 6 ～8×10 6 Putting the individual cells into a centrifuge tube, centrifuging, removing the culture medium, washing once by PBS, centrifuging again, removing the supernatant, and extracting the cell genome DNA of the centrifuged solid phase substance;
2. breaking the genomic DNA extracted in the step one into 500bp fragments, and then purifying by using DNA purification magnetic beads;
in the second step, DNA breaking is carried out by using an instrument Bioraptor, and parameters are as follows: 50 ng/. Mu.L of DNA; the volume is 100 mu L;15sON-90sOFF;6-8 times of circulation; the cleaved DNA was purified with DNA purification beads, and the elution volume was 37. Mu.L.
3. Carrying out end repairing, tail adding and stem-loop structure joint 1 adding on the DNA purified in the step two, then adopting exonuclease treatment, and then using ddNTP for treatment;
in the third step, a kit is adopted for DNA end repair, A tail addition and stem-loop structure joint 1 addition, wherein the specific reaction system and reaction program of the DNA end repair and the A tail addition are as follows:
the kit comprises: the ABClonal rapid DNA library construction kit is purchased from Ebolatukee, wuhan;
reaction procedures are as follows: 30min at 20 ℃; 30min at 65 ℃ to obtain a terminal repairing mixture;
the specific reaction system and reaction procedure of the stem-loop structure-added joint 1 are as follows:
reaction procedures are as follows: after 1h at 22 ℃ the beads were purified 1 XDNA purification, eluting in a volume of 30. Mu.L.
The reaction system and procedure of exonuclease digestion in step three are as follows:
reaction procedures are as follows: 2h at 37 ℃; after 10min at 75 ℃ the column was purified with 1 XDNA purification beads, eluting in a volume of 44. Mu.L.
Reaction system and procedure for ddNTP treatment in step three:
reaction procedures are as follows: at 75 ℃ for 30min; then using 1 x DNA purification magnetic beads purification, elution volume 25 u L, the Qubit measurement of DNA concentration >6.4 ng/. Mu.L.
4. Designing and synthesizing a sgRNA oligo library, and carrying out PCR amplification and in vitro transcription to obtain a sgRNA pool;
in the reaction system and procedure for in vitro cutting of the Cas enzyme in the fifth step, sgRNA oligo pool is firstly designed and synthesized, and after PCR amplification, in vitro transcription is carried out to obtain active sgRNA pool, and the rest is as follows:
reaction procedures are as follows: binding for 10min at room temperature, adding 250ng of DNA treated in the third step, filling the volume with enzyme-free water to 50 μ L, incubating at 37 ℃ for 1h, purifying with 50 μ L DNA purified magnetic beads, and eluting with 37 μ L volume.
5. Carrying out in-vitro cutting on the DNA treated by the ddNTP in the step 3 by using Cas enzyme and sgRNA pool obtained in the step four;
6. carrying out end repairing, tail A adding and linear joint 2 adding on the DNA cut in vitro, wherein the 5' end of the linear joint 2 is modified by biotin;
in the sixth step, an ABClonal rapid DNA library building kit is adopted for DNA end repair, A tail addition and linear joint 2 addition, wherein the ABClonal rapid DNA library building kit is purchased from Ebolatka in Wuhan province; the specific reaction system and reaction program for DNA end repair and A tail addition are as follows:
reaction procedure: reacting at 20 ℃ for 30min; reacting at 65 ℃ for 30min to obtain a terminal repairing mixture;
reaction procedure: after reaction at 22 ℃ for 1h, the column was purified using 1 XDNA purification beads, and the elution volume was 41. Mu.L.
7. Resuspending streptavidin magnetic beads, adding 1 xBind and wash buffer, rotating, uniformly mixing and washing at room temperature, centrifuging, removing supernatant, repeatedly washing, centrifuging and removing supernatant for 3 times, resuspending the streptavidin magnetic beads by using 2 xBind and wash buffer, adding DNA cut in the fifth step, rotating and uniformly mixing at room temperature, placing in a magnetic frame, and removing supernatant to obtain DNA adsorbed by the streptavidin magnetic beads;
the method for treating the USER enzyme in the seventh step comprises the steps of adding the USER enzyme into the DNA in the fifth step, reacting for 30min at 37 ℃, then placing the DNA in a magnetic frame for 2min, removing the supernatant, adding 10mM Tris-HCl, rotating, uniformly mixing and washing for 5min at room temperature, placing the DNA in the magnetic frame for 2min, removing the supernatant, and washing for 2 times by using 10mM Tris-HCl in total; then 20. Mu.L of 10mM Tris-HCl was added for resuspension.
8. Treating DNA adsorbed by streptavidin magnetic beads by using USER enzyme, cutting a stem-loop structure of the joint 1, then performing recovery PCR amplification by using the DNA treated by the USER enzyme as a template, then performing index PCR to obtain a library to be loaded on the computer, and performing library quality inspection and sequencing;
and the recovery PCR reaction system and the reaction program in the step eight are as follows:
reaction procedures are as follows: 45s at 98 ℃;12cycles of (98 ℃ 15s,61 ℃ 30s,72 ℃ 2 min), 72 ℃ 2min,4 ℃ hold, then placed in magnetic rack 2min, the supernatant transferred to new Ep tube, 3u L supernatant diluted 50 times with water.
The reaction system and procedure for indexing the PCR in step eight are as follows:
reaction procedure: 45s at 98 ℃;12cycles of (98 ℃ 15s,60 ℃ 30s,72 ℃ 2 min), 72 ℃ 2min,4 ℃ hold, 0.7 XDNA purification magnetic bead purification, elution volume of 50. Mu.L, 0.6X-0.2 XDNA purification magnetic bead double-end screening, elution volume of 20. Mu.L, get 200-600bp DNA library.
9. And after the library is downloaded, bioinformatics analysis is carried out, each off-target site obtained by bioinformatics analysis is compared back to each sgRNA, and in-target reads and off-target sites corresponding to each sgRNA, the number of the off-target sites and corresponding reads information are obtained.
And (3) testing: as shown in fig. 2 to 4, in order to simultaneously detect data of 3 sites of 74230 sgrnas of human genome by using sgRNA pool strategy, the uppermost base is a target sequence, and the rightmost digit is the number of reads corresponding to each target and off-target site. The more reads, the more cutting.
Claims (8)
1. A method for batch in-vitro off-target detection and sgRNA screening is characterized by comprising the following steps:
1. collecting 4X 10 6 ~8×10 6 Putting the individual cells into a centrifuge tube, centrifuging, removing a culture medium, washing once by PBS, centrifuging again, removing a supernatant, and extracting cell genome DNA of a centrifuged solid-phase substance;
2. breaking the genome DNA extracted in the step one into 300bp to 700bp fragments, and then purifying by using DNA purification magnetic beads;
3. carrying out end repairing, tail adding and stem-loop structure joint adding on the DNA purified in the step two, then adopting exonuclease treatment, and then using ddNTP treatment;
reaction system and procedure for ddNTP treatment in step three:
Reaction procedures are as follows: at 75 ℃ for 30min; then using 1 XDNA purification magnetic beads purification, elution volume 25L, the Qubit measurement DNA concentration >6.4 ng/. Mu.L;
4. designing and synthesizing an sgRNA oligo library, and carrying out PCR amplification and in vitro transcription to obtain sgRNA pool;
5. carrying out in-vitro cutting on the DNA treated by the ddNTP in the step three by using Cas enzyme and sgRNA pool obtained in the step four;
6. carrying out end repairing, tail A adding and linear joint adding on the DNA cut in vitro, wherein the 5' end of the linear joint is modified by biotin;
in the sixth step, an ABClonal rapid DNA library building kit is adopted for DNA end repair, A tail addition and linear joint addition; the specific reaction system and reaction program for DNA end repair and A tail addition are as follows:
Reaction procedure: reacting at 20 ℃ for 30min; reacting for 30min at 65 ℃ to obtain a terminal repairing mixture;
Reaction procedures are as follows: reacting at 22 ℃ for 1h, then purifying by using 1 XDNA purification magnetic beads, and eluting with 41 mu L of volume;
7. resuspending streptavidin magnetic beads, adding 1 xBind and wash buffer, rotating, uniformly mixing and washing at room temperature, centrifuging, removing supernatant, repeatedly washing, centrifuging and removing supernatant for 3 times, resuspending the streptavidin magnetic beads by using 2 xBind and wash buffer, adding DNA cut in the sixth step, rotating and uniformly mixing at room temperature, placing in a magnetic frame, and removing supernatant to obtain DNA adsorbed by the streptavidin magnetic beads;
8. treating DNA adsorbed by streptavidin magnetic beads by using USER enzyme, cutting a stem-loop structure of a joint, then taking the DNA treated by the USER enzyme as a template, performing recovery PCR amplification, then performing index PCR to obtain a library to be loaded, and performing library quality inspection and sequencing;
9. and after the library is downloaded, bioinformatics analysis is carried out, each off-target site obtained by bioinformatics analysis is compared with each sgRNA, and in-target reads and off-target sites corresponding to each sgRNA, the number of the off-target sites and corresponding reads information are obtained.
2. The method of claim 1, wherein in the second step, an instrument Bioraptor is used for DNA disruption, and parameters are as follows: 50 ng/. Mu.L of DNA; the volume is 100 mu L;15sON-90sOFF;6-8 times of circulation; the cleaved DNA was purified with DNA purification beads, and the elution volume was 37. Mu.L.
3. The method of claim 1, wherein in the third step, a kit is used for DNA end repair, A tail addition, and stem-loop structure joint addition, wherein specific reaction systems and reaction procedures for DNA end repair and A tail addition are as follows:
the kit comprises: ABClonal rapid DNA library building kit
Reaction procedure: 30min at 20 ℃; obtaining a terminal repairing mixture at 65 ℃ for 30min;
the specific reaction system and reaction procedure for adding the stem-loop structure joint are as follows:
Reaction procedure: after 1h at 22 ℃ the beads were purified 1 XDNA purification, eluting in a volume of 30. Mu.L.
4. The method for batch in-vitro off-target detection and sgRNA screening according to claim 1, characterized in that the reaction system and procedures of exonuclease digestion in the third step are as follows:
Reaction procedure: 2h at 37 ℃; after 10min at 75 ℃ the column was purified with 1 XDNA purification beads, eluting in a volume of 44. Mu.L.
5. The method of claim 1, which is capable of batch in-vitro off-target detection and sgRNA screening, and is characterized in that in step five, a reaction system for Cas enzyme in-vitro cleavage is as follows:
The reaction procedure was as follows: combining for 10min at room temperature, then adding the DNA 50ng-1000ng processed in the third step, supplementing the volume to 50 mu L with enzyme-free water, incubating for 1h at 37 ℃, purifying by using 50 mu LDNA purified magnetic beads, and eluting the volume to 37 mu L.
6. The method of claim 1, wherein the USER enzyme treatment in step eight is performed by adding the USER enzyme to the DNA in step seven, reacting at 37 ℃ for 30min, placing the DNA in a magnetic rack for 2min, removing the supernatant, adding 10mM Tris-HCl, washing the DNA for 5min at room temperature by rotary mixing, placing the DNA in a magnetic rack for 2min, removing the supernatant, and washing the DNA for 2 times with 10mM Tris-HCl; then 20. Mu.L of 10mM Tris-HCl was added for resuspension.
7. The method of claim 1, wherein the recovery PCR reaction system and the reaction procedure in step eight are as follows:
Reaction procedure: 45s at 98 ℃;12cycles of (98 ℃ 15s,61 ℃ 30s,72 ℃ 2 min), 72 ℃ 2min,4 ℃ hold, then place on magnetic frame for 2min, transfer supernatant to new Ep tube, take 1 uL-10 uL supernatant and add water to dilute 50 times.
8. The method of claim 1, wherein the reaction system and procedures for indexing PCR in step eight are as follows:
Reaction procedure: 45s at 98 ℃;12cycles of (98 ℃ 15s,60 ℃ 30s,72 ℃ 2 min), 72 ℃ 2min,4 ℃ hold, 0.7 XDNA purification magnetic bead purification, elution volume of 50. Mu.L, 0.6X-0.2 XDNA purification magnetic bead double-ended screening, elution volume of 20. Mu.L, get 200-600bp DNA library.
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| Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq;Cicera R Lazzarotto等;《Nat Protoc》;20181130;第13卷(第11期);第2615-1642页 * |
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