A pairing sgRNA for gene editing and its applications

Abstract

This invention discloses a paired sgRNA for gene editing and its applications. Multiple sgRNAs are designed according to the specific editing requirements of the target gene or target genomic locus, and two sgRNAs with a specific spacing and corresponding to a specific Watson / Crick position combination in PAM are selected as paired sgRNAs. The paired Cas9-sgRNA of this invention can improve gene editing efficiency and precision, and can be used to quantitatively analyze NHEJ in cells and animals, including total NHEJ efficiency (i.e., total editing efficiency), the frequency and ratio of precise NHEJ and mutant NHEJ, the direction, length, and frequency of addition / deletion at mutant NHEJ interfaces, and the utilization of microhomologous sequences at mutant NHEJ interfaces. This technology is more flexible, simple, and rapid, and can be easily applied to various cells and tissues without affecting accuracy.

Description

[0001] This application is a divisional application of the invention patent application with application number 201811088469X and application date of September 18, 2018. Technical Field

[0002] This invention relates to a gene editing method, and more particularly to a method for improving the efficiency and accuracy of CRISPR / Cas9 gene editing based on the precise deletion of DNA fragments of specific lengths by manipulating a highly efficient and precise non-homologous end joining (NHEJ) repair pathway. Background Technology

[0003] Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) is currently the most widely used genome editing system. It is derived from the immune mechanisms of bacteria and archaea that degrade invading viral DNA or other exogenous DNA. The most commonly used system is CRISPR / Cas9, which consists of three components: Cas9 protein, crRNA (CRISPR-associated RNA), and tracrRNA (trans-activating crRNA). The Cas9 protein first binds to crRNA and tracrRNA to form a complex. Then, by recognizing and binding to the PAM (protospacer adjacent motif) of the target DNA (e.g., the PAM corresponding to Streptococcus pyogenes SpCas9 is mainly 5'-NGG-3'), it unwinds the target DNA. The small guide RNA (gRNA) containing 20 bases in the crRNA pairs with the single-stranded target DNA bases to form an RNA-DNA complex. Then, the Cas9RuvC and HNH nuclease domains each cleave one strand of the target DNA, forming a double-stranded DNA break with blunt ends. During system optimization, crRNA and tracrRNA can also be fused into a single guide RNA (sgRNA). This sgRNA is simpler and equally effective in mediating Cas9 target cleavage.

[0004] In eukaryotic cells, including human cells, Cas9-induced DNA double-strand breaks are primarily repaired through two pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). DNA double-strand break repair is the theoretical basis for gene editing. HR is a precise repair mechanism, mainly occurring in the G2 and S phases of the cell cycle, utilizing homologous sequences for precise repair. NHEJ occurs throughout the entire cell cycle and is considered an error-prone repair mechanism, during which a certain proportion of base deletions or insertions can occur. The theoretical basis for rapid and efficient gene editing using the CRISPR-Cas9 system lies precisely in the frequent DNA double-strand break repair events resulting from its high DNA cutting efficiency.

[0005] A traditional gene editing procedure utilizes non-precise NHEJ repair to generate indels for gene knockout, deletion of specific DNA fragments, and gene fragment replacement. Generally, higher non-precise NHEJ efficiency suggests higher editing efficiency. However, this isn't always the case. For example, if non-precise NHEJ primarily generates full-frame indels, even high non-precise NHEJ efficiency doesn't necessarily translate to high gene knockout efficiency based on frameshift mutations. Furthermore, non-precise NHEJ products exhibit high heterogeneity, making them difficult to predict and control, unsuitable for precise editing requirements. However, by simultaneously inducing two neighboring DSBs at 71 sites in the genome using paired Cas9-sgRNAs, our analysis of their repair revealed that nearly half of the Cas9-induced NHEJs were precise ligations. This also demonstrates that templated insertions during NHEJ repair affect the efficiency of precise NHEJs. On one hand, this corrects our previous misconception that Cas9-induced DSB NHEJ repair is error-prone. In fact, this result indicates that because the blunt ends of DSBs induced by Cas9 can be directly joined, repair mainly relies on precise NHEJ. Clearly, this inherent precise NHEJ repair is not conducive to mutation generation, hindering CRISPR / Cas9-mediated mutation-based gene editing (including gene knockout). On the other hand, this feature provides an opportunity for gene editing improvement. By inducing adjacent paired DSBs using paired Cas9-sgRNAs and manipulating precise NHEJ repair, we can achieve efficient and controllable precise deletion and insertion, enabling precise and efficient gene knockout, whole-frame deletion, deletion of specific genomic segments, and knock-in or replacement of gene segments. Summary of the Invention

[0006] The purpose of this invention is to provide a method for improving the efficiency and accuracy of CRISPR / Cas9 gene editing by manipulating a highly efficient and precise non-homologous end-joining (NHEJ) repair pathway. Traditional CRISPR / Cas9 gene editing utilizes non-precise NHEJ repair to generate insertions and deletions (i.e., indels) for gene knockout (frameshift mutations), deletion of specific DNA fragments, or gene knock-in (gene fragment replacement), but this method is inefficient, prone to mutations, and exhibits high heterogeneity. This invention's method generates two adjacent double-strand breaks (DSBs) by controlling the spacing and PAM orientation of paired Cas9-sgRNAs at the target site, fully utilizing high-frequency precise NHEJ repair to overcome the influence of templated insertion and efficiently produce precise gene editing products. These products can be, as needed, including frameshift-based gene knockout, full-frame precise deletion, other applications utilizing precise-length genomic fragment deletion, and precise genomic fragment replacement including gene knock-in. This invention also provides an in vivo and in vitro NHEJ analysis method that can replace the NHEJ reporter system, offering flexibility, convenience, and accuracy.

[0007] The technical solution adopted in this invention is: This invention provides a pairing sgRNA for gene editing, wherein the pairing sgRNA is obtained by controlling the spacing and / or corresponding PAM direction of the pairing sgRNA at the target site. That is, multiple sgRNAs are designed according to the target gene or target genomic site, and two sgRNAs with different spacings are selected as pairing sgRNAs according to the gene editing requirements. When the gene editing is a whole-frame deletion, the cutting spacing mediated by the pairing sgRNA is 3n base pairs. When the gene editing is a frameshift mutation or gene knockout, the cutting spacing mediated by the pairing sgRNA is 3n+1 or 3n+2 base pairs, where n is an integer of 0 or above.

[0008] The position of paired Cas9-sgRNAs at the target site is determined by the PAM motif. Cas9-mediated cleavage occurs at the third nucleotide upstream of the 5' end of the PAM, creating a blunt end. The spacing refers to the distance between the cleavage sites of paired Cas9-sgRNAs at the target site. Therefore, the spacing is controlled by selecting the paired PAM according to the editing objective. According to our tests, controlling this spacing between 12-148 base pairs does not reduce the efficiency of precise NHEJ, making it suitable for precise editing of a specific length within a typical exon. Since the PAM motif can be located on either the Watson strand (W) or the Crick strand (C), the arrangement of paired Cas9-sgRNAs at the target site can be combined in four ways based on the PAM positions: W / W, W / C, C / W, and C / C. Due to the wide applicability of PAMs and the recent expansion of PAM compatibility (e.g., from NGG to NG, GAA, and GAT), the target sites to be edited typically contain combinations of these four PAM positions, and each combination offers multiple options. Therefore, we can relatively easily choose one of the combinations to meet specific genome editing needs, with W / C being the preferred option.

[0009] This invention also provides an application of the paired sgRNA in improving the efficiency and accuracy of CRISPR / Cas9 gene editing. The method of application is as follows: multiple sgRNAs are designed according to the target gene, and two sgRNAs with a spacing of 3n, 3n+1, or 3n+2 base pairs are selected as paired sgRNAs according to the gene editing requirements. An sgRNA expression plasmid is constructed and introduced into cells together with a Cas9 expression plasmid for expression. The target DNA is amplified by PCR, and the working efficiency of the paired Cas9-sgRNA is tested by DNA electrophoresis. When the gene editing is a whole-frame deletion, the cleavage spacing mediated by the paired sgRNA is 3n base pairs. When the gene editing is a frameshift mutation or gene knockout, the cleavage spacing mediated by the paired sgRNA is 3n+1 or 3n+2 base pairs, where n is an integer of 0 or above.

[0010] Furthermore, when the paired sgRNA performs frameshift mutations or gene knockouts on the target gene or target genomic site, the position combination is W / C, and the spacing between the paired sgRNAs is 3n+1 or 3n+2 base pairs; the position combination is one of W / W, C / W, or C / C, and the spacing between the paired sgRNAs is 3n+2 base pairs (avoiding 3n+1 base pairs; the basis is that the high-frequency template insertion of a 1-nucleotide can transform a 3n+1 base pair deletion into an entire frameshift deletion, while a 3n+2 base pair deletion, even if transformed into a 3n+1 base pair deletion, is still a frameshift mutation).

[0011] Furthermore, when the paired sgRNAs are subjected to whole-frame mutations or precise whole-frame deletions at target gene or target genomic sites, the spacing between the paired sgRNAs is 3n base pairs, and the positional combination is W / C.

[0012] This invention also provides an application of the paired sgRNA in the quantification of non-homologous end joining in cells or animals. The non-homologous end joining is either precise non-homologous end joining or mutant non-homologous end joining. The application method follows these steps: Paired sgRNAs are designed based on the target gene; sgRNA expression plasmids are constructed and co-introduced into cells with a Cas9 expression plasmid for expression; target DNA is amplified by PCR, and the working efficiency of the paired Cas9-sgRNA is tested by DNA electrophoresis; well-performing paired sgRNAs are selected, and corresponding sgRNA expression plasmids and Cas9 expression plasmids are constructed and co-introduced into cells or tissues for expression; target DNA is amplified by PCR, and NHEJ analysis is performed using deep sequencing PCR amplicon analysis, including total NHEJ efficiency (i.e., total editing efficiency), the frequency and ratio of precise and mutant NHEJs, the insertion / deletion direction, length, and frequency of mutant NHEJ interfaces, and the utilization of micro-homologous sequences of mutant NHEJ interfaces, thereby quantifying NHEJs in cells or animals.

[0013] This invention provides a method for designing paired sgRNAs for precise gene editing, wherein the paired sgRNA positions are combined in the form of W / C, where W represents PAM on the Watson strand and C represents PAM on the Crick strand.

[0014] Preferably, when the purpose of gene editing is to perform frameshift mutations or gene knockouts at target genes or target genomic sites, the spacing between paired sgRNAs is 3n+1 or 3n+2 base pairs; where n is an integer.

[0015] Preferably, when the gene editing objective is to perform full-code mutation or precise full-code deletion on the target gene or target genomic site, the spacing between paired sgRNAs is 3n base pairs; where n is an integer.

[0016] This invention provides a pairing Cas9-sgRNA gene editing method based on precise NHEJ repair, characterized in that the method is as follows: (1) when the pairing sgRNA performs frameshift mutation or gene knockout on the target gene or target genomic site, the pairing sgRNA position combination is W / C, and the spacing is 3n+1 or 3n+2 base pairs; (2) when the pairing sgRNA performs whole-frame mutation or precise whole-frame deletion on the target gene or target genomic site, the pairing sgRNA position combination is W / C, and the spacing is 3n base pairs.

[0017] Preferably, the method operates as follows: multiple sgRNAs are designed based on the target gene, and two sgRNAs with a spacing of 3n or non-3n base pairs are selected as paired sgRNAs according to gene editing requirements, with a PAM position combination of W / C. An sgRNA expression plasmid is constructed and co-introduced into cells with a Cas9 expression plasmid for expression. Target DNA is amplified by PCR, and the efficiency of the paired Cas9-sgRNAs is detected by DNA electrophoresis. The highly efficient W / C paired sgRNA is selected for gene editing. The efficiency of the W / C paired sgRNAs in the gene editing method of this invention can be pre-tested, helping to select the most suitable W / C paired sgRNAs for genome editing. The testing method is as follows: Design multiple sgRNAs based on the target gene, and select two sgRNAs with a spacing of 3n, 3n+1, or 3n+2 base pairs and a PAM position combination of W / C (other combinations are controls) as paired sgRNAs according to the editing requirements. Construct sgRNA expression plasmids, and introduce them into cells together with Cas9 expression plasmids for expression. Amplify the target DNA by PCR, and test the working efficiency of paired Cas9-sgRNAs by DNA electrophoresis.

[0018] Gene segment deletion, including gene knockout, full-column deletion (such as full-column deletion of gene segments encoding functional structural domains), and gene segment replacement after deletion, is an important application of CRISPR gene editing. However, improving the efficiency and accuracy of these applications faces some technical challenges. Currently, CRISPR gene knockout technology mainly utilizes non-precise NHEJ repair to generate base additions and deletions, causing frameshift mutations. However, we have found that the NHEJ repair pathway of Cas9-induced DSBs is mainly precise NHEJ. Obviously, this inherent precise NHEJ repair is not conducive to mutation generation, hindering CRISPR / Cas9-mediated gene knockout based on frameshift mutations. Moreover, theoretically, non-precise NHEJ repair has a one-third probability of generating full-column mutations, and full-column mutations may only affect part of the gene's function, not complete gene knockout. In full-column deletion applications, not only is precise NHEJ unfavorable to full-column mutations, but the probability of full-column additions and deletions in non-precise NHEJ is theoretically only one-third, or 33.3%, resulting in low efficiency. Furthermore, in gene knockout, whole-code deletion, and gene fragment replacement, the lengths of base additions and deletions resulting from non-precise NHEJ repair vary, leading to high heterogeneity of gene editing products that are difficult to predict and control, thus failing to meet the requirements of precise editing.

[0019] Therefore, in order to improve the efficiency and accuracy of gene fragment deletion applications, we have invented a method to efficiently generate two adjacent DSBs by manipulating the spacing and orientation of paired Cas9-sgRNAs at the target site. This method utilizes high-frequency, precise NHEJ repair to overcome the interference of templated insertion, efficiently generating precise gene editing products and achieving efficient and precise gene editing, including gene knockout and whole-code deletion (such as whole-code deletion of gene fragments encoding functional structural domains). Specific problems to be solved by this invention include: (1) the selection of the spacing and orientation of paired sgRNAs at the target site; (2) the prediction of paired sgRNA-guided cleavage efficiency; (3) improving gene knockout efficiency and reducing the heterogeneity of knockout products; and (4) improving the efficiency and homogeneity of whole-code deletion of specific endogenous gene fragments (applications include precise deletion of gene fragments encoding specific structural domains or specific functional motifs). Due to the efficient and precise NHEJ repair, we hypothesize that the efficiency of precise genome fragment replacement (including gene knock-in) can also be improved by manipulating accurate NHEJ repair.

[0020] The NHEJ reporter system is a primary method for quantitative analysis of NHEJs in vivo and in vitro. However, it has several limitations, such as measurement restriction to reporter site, difficulty in integration into heterochromatin, and the time-consuming and labor-intensive process of establishing an NHEJ reporter system in animals. This invention provides an alternative in vivo and in vitro NHEJ analysis technique that replaces the traditional reporter system. Through target PCR amplicon next-generation sequencing and bioinformatics analysis, it can distinguish and quantify precise and mutant NHEJs in vivo or in vitro, determine NHEJ repair efficiency and patterns, and is more flexible, convenient, and accurate. It can be used not only to analyze euchromatin NHEJs in cells, tissues, and organs but also to analyze heterochromatin NHEJs in cells, tissues, and organs. Therefore, this invention also provides a flexible, rapid, and accurate method for detecting and quantifying intracellular and in vivo non-homologous end joins (NHLs). This method does not rely on traditional reporter systems and can distinguish between precise and mutant NHLs.

[0021] Compared with existing technologies, the paired Cas9-sgRNA gene editing technology of this invention has the following advantages: This invention proposes a W / C paired sgRNA combination that does not produce templated insertion, resulting in higher precision NHEJ efficiency than other combinations. Utilizing the W / C paired Cas9-sgRNA editing technology of this invention, frameshift mutation gene knockout can be achieved by pre-setting the spacing between the sgRNAs at the target site to 3n+1 or 3n+2 base pairs. This editing produces a high proportion of precisely deleted gene products of a specific length, with improved homogeneity, facilitating the acquisition of the desired gene editing product. For more challenging applications of precise whole-frame deletion gene editing, this invention proposes a W / C paired sgRNA spacing of 3n base pairs, enabling gene editing of specific gene fragments with high homogeneity.

[0022] The new technical solution proposed in this invention is flexible, simple, and rapid, and can be easily applied to various cells and tissues with high accuracy. It can also measure and analyze NHEJ in primary cells isolated from clinical patients, which is of great value for future clinical assessment of patients' NHEJ repair capacity and for guiding physicians in selecting radiotherapy and chemotherapy dosages. Attached Figure Description

[0023] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this specification and, together with the description, serve to explain the principles of this specification.

[0024] Figure 1 Comparison of paired Cas9-sgRNA NHEJ analysis technology with I-SceI-induced NHEJ. a. Schematic diagram of the working patterns of I-SceI and paired Cas9-sgRNA on the NHEJ reporter system (sGEJ). b. Comparison of the efficiency of I-SceI and paired Cas9-sgRNA-induced NHEJ. c. Proportional distribution of the four groups of NHEJ induced by I-SceI and paired Cas9-sgRNA. d. Distribution of precise and mutant NHEJs in Group I NHEJ induced by I-SceI and paired Cas9-sgRNA.

[0025] Figure 2 Analysis of NHEJ at 71 endogenous genomic loci using paired Cas9-sgRNA technology. a. Schematic diagram of NHEJ products (genome editing) induced by paired Cas9-sgRNA. b. Overall editing efficiency, Group I NHEJ efficiency, and precision NHEJ efficiency at 71 loci. c. Loci with low precision NHEJ efficiency are often accompanied by high-frequency base insertions.

[0026] Figure 3 The impact of templated insertion at 71 genomic loci on the efficiency of precise NHEJ. a. Relationship between insertion efficiency and templated insertion efficiency. This figure shows that almost all insertions are templated insertions. b. Templated insertion efficiency of high-frequency precise NHEJs and 1-nucleotide and 2-nucleotide insertions in NHEJ products. c. Schematic diagram showing the negative correlation between 1-nucleotide and 2-nucleotide templated insertion efficiency and precise NHEJ efficiency.

[0027] Figure 4 : Schematic diagram predicting the impact of PAM combinations paired with Cas9-sgRNA on templated insertion. Based on this diagram, it is inferred that W / C PAM combinations paired with Cas9-sgRNA will not produce templated insertion.

[0028] Figure 5The effect of PAM combinations paired with Cas9-sgRNA on templated insertion. a. Schematic diagram of the effect of four PAM combinations paired with Cas9-sgRNA on templated insertion efficiency. This figure shows that the W / C combination does not produce templated insertion. b. Schematic diagram of the interference of templated insertion on the precise NHEJ frequency in W / W, W / C, C / W, and C / C. This figure shows that the interference of templated insertion results in a lower precise NHEJ frequency in W / W, C / W, and C / C than in W / C.

[0029] Figure 6 The effect of Cas9-sgRNA pairing spacing on the efficiency of precise NHEJ. a. Schematic diagram of the effect of Cas9-sgRNA pairing spacing on the efficiency of precise NHEJ. This figure shows that the Cas9-sgRNA pairing spacing has no effect on the efficiency of precise NHEJ. b. Schematic diagram of the effect of W / W combination Cas9-sgRNA pairing spacing on the efficiency of precise NHEJ. This figure shows that the W / W combination Cas9-sgRNA pairing spacing has no effect on the efficiency of precise NHEJ.

[0030] Figure 7 : Paired Cas9-sgRNA cleavage efficiency test and screening and editing of highly efficient paired sgRNAs. a. Schematic diagram of human AAVS1 paired sgRNA design and targeting. b. Schematic diagram of human HBB paired sgRNA design and targeting. c. DNA electrophoresis analysis of human AAVS1 and HBB paired sgRNA efficiency test and screening.

[0031] Figure 8 Paired Cas9-sgRNA technology can improve frameshift-based gene knockout editing. a. Schematic diagram of paired Cas9-sgRNA editing product grouping. Paired Cas9-sgRNA editing products can be divided into three groups: "Ideal," "Common," and "Paired." GI, GII, GIII, and GIV represent Groups I, II, III, and IV, respectively. b. Schematic diagram of the correlation analysis between precise NHEJ frequency and frameshift mutation efficiency. This graph shows a positive correlation between precise NHEJ frequency and frameshift mutation efficiency. c. Schematic diagram of the correlation between 1nt templated insertion and frameshift mutation efficiency. This graph shows a negative correlation between 1nt templated insertion and frameshift mutation frequency of "3n+1" base pair deletion instead of "3n+2" base pair deletion. d. Relationship between "3n+1" and "3n+2" base pair deletion and frameshift mutation efficiency. The figure shows that, on average, “3n+2” base pair deletions based on the precise NHEJ, instead of “3n+1” base pair deletions, can increase the frameshift mutation frequency.

[0032] Figure 9Paired Cas9-sgRNA technology can improve whole-column mutation editing. a. Comparison of paired strategies with other strategies in whole-column mutation. This figure shows that paired strategies are more conducive to improving whole-column mutation efficiency than traditional strategies. b. Schematic diagram showing a positive correlation between whole-column mutation efficiency and accuracy NHEJ efficiency.

[0033] Figure 10 Tests using paired Cas9-sgRNA NHEJ analysis technology. a. Schematic diagram of the effect of XRCC4 deletion on overall NHEJ efficiency (gene editing efficiency). LDHA and ROSA26 are two test targets. b. Schematic diagram of the effect of XRCC4 deletion on the four categories of NHEJ products. c. Schematic diagram of the effect of XRCC4 deletion on the efficiency of precise and mutant NHEJ.

[0034] Figure 11 Schematic diagram of the impact of XRCC4 deletion on NHEJ interface deletion length (a) and microhomology utilization (b). MH: microhomology.

[0035] Figure 12 : Testing of paired Cas9-sgRNA NHEJ analysis technology in mouse liver. a. Flowchart of injection of plasmids expressing paired Cas9-sgRNA targeting LDHA into mice via hydrodynamic tail vein injection. Liver tissue was collected 30 days after injection, genomic DNA was purified, target amplification was performed by PCR, and deep sequencing analysis was conducted to determine the overall editing efficiency, Group I-IV NHEJ efficiency distribution, and the distribution of precise NHEJs and mutant NHEJs (Del, Ins, and InDel) for the same LDHA target. b. Schematic diagram of editing efficiency. c. Group I-IV NHEJ efficiency distribution. d. Distribution of precise NHEJs and mutant NHEJs (Del, Ins, and InDel). H211, H221, H521, and H531 are four liver tissue samples, and n refers to the number of next-generation sequencing sequences. NS: not significant.

[0036] Figure 13 Analysis of NHEJ in mouse liver. a. Schematic diagram of deletion length atlas of NHEJ in mouse liver. b. Relationship between NHEJ microhomological sequence utilization and length. H211, H221, H521, and H531 are four liver tissue samples, and n refers to the number of next-generation sequencing sequences.

[0037] Figure 14 Flowchart of paired Cas9-sgRNA gene editing.

[0038] Figure 15: MDC1 gene knockout using paired Cas9-sgRNA technology. a. DNA electrophoresis analysis of high-efficiency paired sgRNAs targeting MDC1 on exon 2. b. Schematic diagram of the precise NHEJ efficiency and mutation efficiency distribution of paired Cas9-sgRNAs. c. Table of single-clone screening results. d. Western blotting plot validating the MDC1 knockout results.

[0039] Figure 16 : Paired Cas9-sgRNA technology for full-frame precise deletion of the gene segment encoding the Tudor and OD domains of 53BP1. a. Sanger sequencing map verifying full-frame precise deletion of the OD and Tudor domains. b. Western blotting map verifying unaffected 53BP1 protein expression. c. Focal immunofluorescence map showing the recruitment of 53BP1 to the DSB site by OD and Tudor mutants.

[0040] Figure 17 : Heterochromatin NHEJ analysis based on paired Cas9-gRNA NHEJ analysis technology. Through testing, a pair of paired sgRNAs targeting constitutive heterochromatin targets Misat and TRPC21 were selected. Cas9 and paired sgRNA expression plasmids were transfected into mouse embryonic stem cells, generating paired DSBs at the target sites. After 72 hours, genomic DNA was collected, and PCR amplification was performed on target sequences containing NHEJs for scar repair. Deep sequencing and analysis revealed the NHEJ repair and editing capabilities at each target site. a. Overall editing efficiency diagram. b. Group I-IV NHEJ efficiency distribution diagram. c. Distribution diagram of precise NHEJs and mutant NHEJs (Del, Ins, and InDel).

[0041] Figure 18 Schematic diagram of the deletion length of the mutant NHEJ interface of the constitutive heterochromatin targets Misat and TRPC21 (a) and schematic diagram of the relationship between micro-homologous sequence utilization and length (b). Detailed Implementation

[0042] To make the purpose, technical solution, and advantages of this disclosure clearer, the following detailed description is provided through specific embodiments.

[0043] In this disclosure, the term "PAM (Protospacer Adjacent Motif)" refers to a short DNA sequence located downstream of the DNA region targeted for cleavage by the CRISPR system. It is an essential sequence for Cas nucleases (such as Cas9) to achieve DNA binding and cleavage, and its presence is a key mechanism for the CRISPR system to distinguish itself from foreign DNA and avoid miscutting its own genome.

[0044] In this disclosure, the terms "Watson chain (W chain)" and "Crick chain (C chain)" have opposite meanings and are in an antiparallel relationship; along the upstream to downstream extension direction, the nucleotide arrangement of the Watson chain is 5'→3', while the nucleotide arrangement of the Crick chain is 3'→5'. In the pairing sgRNA position combination form, the first letter represents the chain on which the PAM of the upstream target is located, and the second letter represents the chain on which the PAM of the downstream target is located. "W / C" means that the PAM of the upstream target is on the Watson chain, and the PAM of the downstream target is on the Crick chain.

[0045] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto: Example 1: An improved method for paired Cas9-sgRNA gene editing 1. Establishment of the theoretical framework for a precise NHEJ-based paired Cas9-sgRNA gene editing method.

[0046] In recent years, gene editing technology using Cas9 guided by a single sgRNA has been widely applied, especially for gene knockout based on frameshift mutations. This technology utilizes non-precise NHEJ repair to generate additions and deletions, of which only a portion (theoretically two-thirds) result in a single-allelic frameshift mutation, thus knocking out one allele. However, due to the mixed and varied lengths of the added and deleted base sequences, the NHEJ products exhibit high heterogeneity, leading to low efficiency in simultaneous knockout of two alleles. Furthermore, this application overlooks an important issue: since the ends of the DSBs generated by Cas9 cleavage can be directly joined, NHEJ repair may primarily be precise, thus reducing the efficiency of gene knockout based on frameshift mutations. Gene editing using Cas9 guided by a single sgRNA also struggles to distinguish between precise NHEJs and targets that have not undergone breakage.

[0047] To clarify whether Cas9-induced NHEJ is primarily precise, we used a previously constructed NHEJ reporter system based on paired I-SceI cleavage (Xie et al, Nature Structural & Molecular Biology, 2009, 16: 814-818). We designed paired sgRNAs (gsGEJ2+gsGEJ2a) near the I-SceI recognition site of the reporter system (see nucleotide sequence 56 in the corresponding 71 genomic loci) based on the target sequence. This reporter system contains a GFP gene with an additional strong translation initiation site “Kozak-ATG” (Koz-ATG for short) added before the normal GFP gene's ATG translation initiation site. Koz-ATG has two I-SceI cleavage sites on each side (e.g., ...).Figure 1 (a) Due to the strong translation initiation site "Koz-ATG", GFP translation is initiated by Koz-ATG. At this time, GFP is frameshifted, therefore the cell is GFP-negative. When the cell expresses a rare exogenous I-SceI endonuclease, Koz-ATG is cleaved, producing DSB. If the cell chooses the NHEJ method to repair the DSB, the repaired cell's GFP will lack Koz-ATG, and translation will begin with the normal ATG, thus synthesizing normal GFP protein, and the cell becomes GFP-positive. Similar to I-SceI cleavage, Cas9, guided by paired sgRNA (gsGEJ2+gsGEJ2a), creates a cleavage at the reporter system target site, producing DSB. NHEJ repair will produce four different products, which have the following characteristics at the NHEJ repair interface ( Figure 1 (a) (1) In Group I, both sites are cut simultaneously, resulting in the deletion of the intermediate DNA fragment, and the remaining two ends are linked by NHEJ. If there is no additional base addition or deletion at the two ends when linked, it is a precise NHEJ; otherwise, it is a mutant NHEJ. (2) In Group II, the upstream cleavage site is edited while no editing "traces" are seen at the downstream target site. (3) In Group III, the downstream cleavage site is edited while no editing "traces" are seen at the upstream target site. (4) In Group IV, both cleavage sites are edited independently, and the intermediate DNA fragment is not completely lost. Through target PCR, amplicon deep sequencing and bioinformatics analysis, the characteristics of the DNA sequence at the NHEJ interface can not only quantitatively reflect the total editing efficiency of the total NHEJ efficiency ( Figure 1 (b) can also be used to identify these four different groups of products ( Figure 1 (c), and measured the ratio of precise NHEJ and mutant NHEJ in the first group, as well as the deletions (Del), insertions (Ins), and additions / deletions (InDel) in mutant NHEJ. Figure 1 (d).

[0048] We transfected mouse embryonic stem cells with I-SceI expression plasmids or paired sgRNAs in combination with Cas9 plasmids. Three days after transfection, genomic DNA was extracted from the cells, and the editing target sites were amplified by PCR and deep sequenced to analyze the DNA sequence characteristics of the target NHEJ interface. We found that I-SceI caused DSB in the reporter system at a rate of approximately 10%, which was repaired by NHEJ, while the editing rate of paired Cas9-sgRNA in the reporter system was significantly higher than that of I-SceI, reaching 80%. Figure 1(b). In the reporting system, products that pair Cas9 with the reporting system and directly connect the two DSBs account for more than 70% of all edited products. Figure 1 (c) DSB caused by Cas9 can be repaired by precise NHEJ, with an efficiency exceeding 70% of the first group of NHEJs. Figure 1 (d).

[0049] To further confirm that Cas9-sgRNA-induced NHEJ is primarily precise, we designed arbitrarily paired sgRNAs targeting 71 endogenous genomic loci, including human and mouse genomes, using a common sgRNA design methodology (i.e., sgRNA expression plasmid construction). The Cas9 cleavage sites mediated by these paired sgRNAs were spaced 12-148 base pairs apart. Combined with Cas9 plasmids, mouse embryonic stem cells and human cells were transfected using a universal transfection method. The paired sgRNAs guided Cas9 to induce DSB, and the cells initiated a repair mechanism to perform NHEJ repair on the corresponding DSB.

[0050] Three days after transfection, cells were collected and genomic DNA was extracted. Primers were designed at the targeted editing sites, and amplification was performed by PCR. The PCR amplicon was deep sequenced, and the DNA sequence characteristics at the NHEJ interface of each genomic site were analyzed. The overall editing efficiency, NHEJ efficiency of Groups I, II, III, and IV, and the frequency of precise and inaccurate NHEJ in Group I were calculated using the deep sequencing DNA sequence reads corresponding to each characteristic. Figure 2 (a). Among the NHEJ products from 71 genomic loci, the average gene editing efficiency was approximately 35.8%, with over 60% of the products being Group I NHEJ products. Of these, approximately 50% were precise NHEJs. Figure 2 (b) This result indicates that cells tend to utilize precise NHEJ to repair Cas9-induced DSB, and the efficiency is quite considerable. However, the efficiency of precise NHEJ varies greatly at different sites, reaching over 90% at high sites and only around 10% at low sites. Figure 2 (b) Through analysis of these 71 endogenous genomic loci (see locus information for details) Figure 2 Analysis at each interface (c) revealed that sites with low precision NHEJ efficiency typically exhibit a large number of insertions at the NHEJ interface. Figure 2 (c) Analysis of these inserted sequences revealed that they were mostly 1-nucleotide (1 nt) insertions, and not random, but consistent with the template sequence; we call this templated insertion (TI). Figure 3(a) The reason for templated insertion is that the RuvC domain of Cas9 can not only cleave the third base before the PAM motif to produce a blunt end, but also cleave at the fourth or even fifth base, thus producing a 5' sticky end with a 1-nt or 2-nt extension. This sticky end can be padded to a blunt end by polymerase, resulting in base insertion during ligation, and the sequence is completely identical to the template. We found that the proportion of 1-nt and 2-nt templated insertions in NHEJ induced by paired Cas9-sgRNA was relatively high. Figure 3 (b) It can be inferred that templated insertion reduces the efficiency of precise NHEJ. The results show that the proportion of 1-nt and 2-nt templated insertions is negatively correlated with the efficiency of precise NHEJ; the higher the proportion of templated insertions, the lower the efficiency of precise NHEJ. Figure 3 (c). However, even if a single nick produced by a single Cas9-sgRNA may have a 5' sticky end, precise NHEJ is more likely to occur and templated insertion is prevented because the ends are complementary. Conversely, double nicks with 5' sticky ends produced by paired Cas9-sgRNAs typically have non-complementary ends, making precise NHEJ difficult to perform and templated insertion more likely. Therefore, the precise NHEJ efficiency measured using the paired Cas9-sgRNA strategy underestimates the actual precise NHEJ efficiency used to repair DSBs generated by Cas9 cleavage.

[0051] Nucleotide sequences of 71 genomic loci paired with Cas9-sgRNA editing targets and their corresponding sgRNA target locations (underlined; -NGG PAM or their complementary sequences are marked with uppercase letters): (1) Nucleotide sequence of the Cas9-sgRNA target site at the first locus of ATM on human chromosome 11 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with a capital letter) ggatcttccagagataggctacagattgcaacccaattaatatcaaagtatcctgcaagttta CCTaactgtgagctgtctccatt actgatgatactatctcagcttctaccccaacagcgacatggggaacgtacaccatatgtgttacgatg CCTtacggaagttgcattgtgt caagacaagaggtcaaacctagaaagctcacaaaagtcagatttattaaaactctggaataaaatttggtgtattacctttcgtggtataagttctgagcaaatacaagctgaaaactttggcttacttggagccataattcagggtagtttagttgaggttgacagagaattctggatcgtcgaacggcag.

[0052] (2) Nucleotide sequence of the Cas9-sgRNA target site at the second locus of ATM on human chromosome 11 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) gaaataagtgtgattagtaacccattattatttcctttttatttcagaaagaagttgagaaatttaagcg CCTgattcgagatcctgaaacaa ttaaacatctagatcggcattcagatt CCAaacaaggaaaatatttgaat tgggatgctgtttttaggtattctattcaaatttattttactgtctttatttttctctttcatatttatttctgttg.

[0053] (3) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd locus of ATM on human chromosome 11 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) gaaataagtgtgattagtaacccattattatttcctttttatttcagaaagaagttgagaaatttaagcg CCTgattcgagatcctgaaacaa ttaaacatctagatcggcattcagattcca aacaaggaaaatatttgaatTGG gatgctgtttttaggtattctattcaaatttattttactgtctttatttttctctttcatatttatttctgttg.

[0054] (4) Nucleotide sequence of the Cas9-sgRNA target site paired at the first site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggg gcggccagtttcccgggttCGG ctttacgtca cgcgagggcggcagggaggaCG Gaatggcggggtttggggtgggtccctcctcgggggagccctgggaaaagaggactgcgtgtgggaagagaaggtggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatggcgcggggt.

[0055] (5) Nucleotide sequence of the Cas9-sgRNA target site at the second site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcggggg cggccagtttcccgggttCGG ctttacgtcacgcgagggcggcaggg aggacg gaatggcggggttTGG ggtgggtccctcctcgggggagccctgggaaaagaggactgcgtgtgggaagagaaggtggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0056] (6) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggg gcggccagtttcccgggttCGG ctttacgtcacgcgagggcggcagggaggacggaatggcgggg tttggggtgggtccctcctCGG gggagccctgggaaaagaggactgcgtgtgggaagagaaggtggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0057] (7) Nucleotide sequence of the Cas9-sgRNA target site at the 4th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggg gcggccagtttcccgggttCGG ctttacgtcacgcgagggcggcagggaggacggaatggcggggtttggggtgggtccctcct cgggggagccctgggaaaagAGG actgcgtgtgggaagagaaggtggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0058] (8) Nucleotide sequence of the Cas9-sgRNA target site at the 5th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggg gcggccagtttcccgggttCGG ctttacgtcacgcgagggcggcagggaggacggaatggcggggtttggggtgggtccctcctcggggggagccctgggaaaagag gactgcgtgtgggaagagaAGG tggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0059] (9) Nucleotide sequence of the Cas9-sgRNA target site at the 6th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggggcggccagtttcccgggttcg gctttacgtcacgcgagggCGG caggg aggacg gaatggcggggttTGGggtgggtccctcctcgggggagccctgggaaaagaggactgcgtgtgggaagagaaggtggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0060] (10) Nucleotide sequence of the Cas9-sgRNA target site at the 7th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggggcggccagtttcccgggttcg gctttacgtcacgcgagggCGG cagggaggacggaatggcgggg tttggggtgggtccctcctCGG gggagccctgggaaaagaggactgcgtgtgggaagagaaggtggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0061] (11) Nucleotide sequence of the Cas9-sgRNA target site at the 8th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggggcggccagtttcccgggttcg gctttacgtcacgcgagggCGG cagggaggacggaatggcggggtttggggtgggtccctcct cgggggagccctgggaaaagAGG actgcgtgtgggaagagaaggtggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0062] (12) Nucleotide sequence of the Cas9-sgRNA target site at the 9th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggggcggccagtttcccgggttcg gctttacgtcacgcgagggCGG cagggaggacggaatggcggggtttggggtgggtccctcctcggggggagccctgggaaaagag gactgcgtgtgggaagagaAGG tggaaatggcgttttggttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0063] (13) Nucleotide sequence of the Cas9-sgRNA target site at the 10th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) atccgcagtgcgggctcgggcggccgggcccagggaaccccgcaggcgggggcggccagtttcccgggttcg gctttacgtcacgcgagggCGG cagggaggacggaatggcggggtttggggtgggtccctcctcgggggagccctgggaaaagaggactgcgtgtgggaag agaagg tggaaatggcgtttTGG ttgacatgtgccgcctgcgagcgtgctgcggggaggggccgagggcagattcgggaatgatggcgcggggt.

[0064] (14) Nucleotide sequence of the Cas9-sgRNA target site at the 11th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with a capital letter) actttctattaaattcctgattttatttctgtaggactgaacg tcttgctcgagatgtgatgaAGG agatgggaggccatcacattgtagc CCTctgtgtgct caaggggggctataaattctttgctgacctgctggattacatcaaagcactgaatagaaatagtgatagatccattcctatgactgtagattttatcagactgaagagctattgtgtgagtatatttaatatatgattctttttagtggca.

[0065] (15) Nucleotide sequence of the Cas9-sgRNA target site paired at the 12th site of HPRT on human X chromosome and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) actttctattaaattcctgattttatttctgtaggactgaacgtcttgctcgagatgtgatgaaggagatgggaggccatcacattgtagccctctgtgtgctcaaggggggctataaattctttgctga CCTgctggattacatcaaagcac tgaatagaaatagtgatagat CCAtt cctatgactgtagatttt atcagactgaagagctattgtgtgagtatatttaatatatgattctttttagtggca.

[0066] (16) Nucleotide sequence of the Cas9-sgRNA target site at the first locus on p53 of human chromosome 17 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) cacccatctacagtcccccttgccgtcccaagcaatggatgatttgatgctgtcc CCGgacgatattgaacaatggtt cactgaagacccaggtccagatgaagctcccagaat gccagaggctgctccccccgTGG cccctgcaccagcagctcctacaccggcggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccagaaaacctaccagggcagct.

[0067] (17) Nucleotide sequence of the Cas9-sgRNA target site at the second locus on p53 of human chromosome 17 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) cacccatctacagtcccccttgccgtcccaagcaatggatgatttgatgctgtccccggacgatattgaa caatggttcactgaagacccAGGtccagatgaagctcccagaat gccagaggctgctccccccgTGG cccctgcaccagcagctcctacaccggcggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccagaaaacctaccagggcagct.

[0068] (18) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd locus on p53 of human chromosome 17 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) cacccatctacagtcccccttgccgtcccaagcaatggatgatttgatgctgtccccggacgatattgaa caatggttcactgaagacccAGG tccagatgaagctcccagaatg CCAgaggctgctccccccgtggc ccctgcaccagcagctcctacaccggcggcccctgcaccagccccctcctggcccctgtcatcttctgtcccttcccagaaaacctaccagggcagct.

[0069] (19) Nucleotide sequence of the paired Cas9-sgRNA target site on mouse chromosome 2, 53BP1 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) ttccattccaggggagcagatggaccctactggaagtcaattggattcagatttctctcagcaagacactccttgcctgataatagaagattctcagcctgaaagc c aggttctggaagaagatgcAGG ctctcacttcagcgtgctatctcgaca CCTtcctaatctgcagatgcaca aagagaaccccgtgttggtgagtgagtgatgatgccctgttcaag.

[0070] (20) Nucleotide sequence of Artemis-paired Cas9-sgRNA target site on mouse chromosome 2 and location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgtgcttctgtttccgaggtgggctttgggtctagtgttcccgggtgcgatgag ctccttccagggacagatggCGG agtatccaacca tctccattgaccgctt cgacAGGgagaacctgaaagcccgtgcctacttcctttcgca.

[0071] (21) Nucleotide sequence of the ATM-paired Cas9-sgRNA target site on mouse chromosome 9 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with a capital letter) taggttgatcccacaggagagtatgaaaatctggtgactataaaatcatttaaa acagaatttcgcttagctggAGG cttaaatttacccaaaataataga ttgtgtgg gttctgatggcaAGG aaaggagacagcttgtgaaggtaagacttcctgtttctggtatagtcttcagaggattaatgccatcaacatcaacagcaggatatttaattcttacctcatttggttgtctggat.

[0072] (22) Nucleotide sequence of the target Cas9-sgRNA paired with ATR on mouse chromosome 9 and location of the target sgRNA (underlined; -NGG PAM or its complementary sequence is marked with capital letters) gcaagaatcctgctatttttggggtactcacaagagaattactttatctttttgaagacttaatttacctccacaaaagaaatg cagtgggtgaggttatggaaTGG ccagtggttgttagtcggttttt aagccgattggatgaacataTGG gatgtttacagccagctcctttgcagttcatgaacgtgcaaa.

[0073] (23) Nucleotide sequence of the first site of the paired Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtgggagggcgga CCTggcattaccctgttatccct agaattctgcagtcgacggta CCGcgggcccgggatccatcgcc accatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0074] (24) Nucleotide sequence of the second site paired with Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtgggagggcgga CCTggcattaccctgttatccct agaat tctgcagtcgacg gtaccgcGGG cccgggatccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0075] (25) Nucleotide sequence of the 3rd site paired with Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggt tatgtgtgggagggcggaccTGG cattaccctgttatccctagaattctgcagtcgacggta CCGcgggcccgggatccatcgcc accatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0076] (26) Nucleotide sequence of the 4th site of the paired Cas9-sgRNA target in a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tcggaaatgagaacaggggcatcttgagcccctgcggacggtgccgacagg tgcttctcgatctgcatccTGG gatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtggggagggcggacctggcattaccctgttat ccctagaattctgcagtcgaCGG taccgcgggcccgggatccatcgccaccatggtgagcaagggcgaggagctgttca.

[0077] (27) Nucleotide sequence of the 5th site of the paired Cas9-sgRNA target in a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tcggaaatgagaacaggggcatcttgagcccctgcggacggtgccgacagg tgcttctcgatctgcatccTGG gatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggttatgtgtggggagggcggacctggcattaccctgttatccctagaattctgcag tcgacggtaccgcgggcccGGG atccatcgccaccatggtgagcaagggcgaggagctgttca.

[0078] (28) Nucleotide sequence of the 6th site of the paired Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggt tatgtgtgggagggcggaccTGG cattaccctgttat ccctagaattctgcagtcgaCG G taccgcgggcccgggatccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0079] (29) Nucleotide sequence of the 7th site of the paired Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggt tatgtgtgggagggcggaccTGG cattaccctgttatccctagaat tctgcagtcgacg gtaccgcGGG cccgggatccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0080] (30) Nucleotide sequence of the paired Cas9-sgRNA target site at the 8th site on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctgccctctggt tatgtgtgggagggcggaccTGG cattaccctgttatccctagaattctgcag tcgacg gtaccgcgggcccGGG atccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0081] (31) Nucleotide sequence of the 9th site of the paired Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctg ccctctggttatgtgtgggaGGG cggacctggcattaccctgttat ccctagaattctgcagtcgaCG G taccgcgggcccgggatccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0082] (32) Nucleotide sequence of the 10th site of the paired Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattgctg ccctctggttatgtgtgggaGGG cggacctggcattacctgttatccctagaattctgcag tcgacg gtaccgcgggcccGGG atccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0083] (33) Nucleotide sequence of the 11th site of the paired Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagt gatggacagccgacggcagtTGG gattcgtgaattgctgccctctggttatgtgtgggagggcggacctggcattaccctgttat ccctagaattctgcagtcgaCG G taccgcgggcccgggatccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0084] (34) Nucleotide sequence of the 12th site of the paired Cas9-sgRNA target on a single copy of the BGN reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcttctcgatctgcatcctgggatcaaagccatagtgaaggacagtg atggacagccgacggcagtTGG gattcgtgaattgctgccctctggttatgtgtggggagggcggacctggcattaccctgttatccctagaattctgcag tcgacg gtaccgcgggcccGGG atccatcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctg.

[0085] (35) Nucleotide sequence of the Cas9-sgRNA target site at the first locus on mouse chromosome 11 (Cola1) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) ccactgattgacacgttgattgggtaaaacctttcttcccaggcatca CCAgtcttgggactcttcctggc tagaccctatctcctcccatcctcacagggtccatccttctgaactcagcat CCGagctgtacctggccactgct cgcttgtctgaacttactgtctcctccacgagcccccatctgtcataccagacaaagggtgtgact.

[0086] (36) Nucleotide sequence of the Cas9-sgRNA target site at the second locus on mouse chromosome 11 (Cola1) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) ccactgattgacacgttgattgggtaaaacctttcttcccaggcatca CCAgtcttgggactcttcctggc tagaccctatctcctcccatcctcacagggtccatccttctgaactcagcatccgagctgta CCTggccactgctcgcttgtctg aacttactgtctcctccacgagcccccatctgtcataccagacaaagggtgtgact.

[0087] (37) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd locus on mouse chromosome 11 (Cola1) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) ccactgattgacacgttgattgggtaaaacctttcttcccaggcatc accagtcttgggactcttccTGG ctagaccctatctcctcccatcctcacagggtccatccttctgaactcagcat CCGagctgtacctggccactgct cgcttgtctgaacttactgtctcctccacgagcccccatctgtcataccagacaaagggtgtgac.

[0088] (38) Nucleotide sequence of the Cas9-sgRNA target site at the 4th locus on mouse chromosome 11 (Cola1) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) ccactgattgacacgttgattgggtaaaacctttc ttcccaggcatcaccagtctTGG gactcttcctggctagaccctatctcctcccatcctcacagggtccatccttctgaactcagcat CCGagctgtacctggccactgct cgcttgtctgaacttactgtctcctccacgagcccccatctgtcataccagacaaagggtgtgact.

[0089] (39) Nucleotide sequence of the Cas9-sgRNA target site at the 5th locus on mouse chromosome 11 (Cola1) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with a capital letter) ccactgattgacacgttgattgggtaaaacctttc ttcccaggcatcaccagtctTGG gactcttcctggctagaccctatctcctcccatcctcacagggtccatccttctgaactcagcatccgagctgta CCTggccactgctcgcttgtctg aacttactgtctcctccacgagcccccatctgtcataccagacaaagggtgtgact.

[0090] (40) Nucleotide sequence of the Cas9-sgRNA target site at the 6th locus on mouse chromosome 11 (Cola1) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) ccactgattgacacgttgattgggtaaaacctttcttcccaggcatca CCAgtcttgggactcttcctggc tagaccctatctcctcccatcctcacagggtccatccttctgaa ctcagcatccgagctgtaccTGG ccactgctcgcttgtctgaacttactgtctcctccacgagcccccatctgtcataccagacaaagggtgtgact.

[0091] (41) Nucleotide sequence of the target Cas9-sgRNA paired with Ku80 on mouse chromosome 1 and location of the target sgRNA (underlined; -NGG PAM or its complementary sequence is marked with capital letters) acaccgtcttaaacatgttctctctccctttatcctttttacgtagcaggacagggacccacttcagaaactttgctaatagcgtctttttttcttgtgtacttttggggcctttgttctaggcagctgttgtgctgtg tgtggatgtgggggttgccaTGG gtaactccttt CCTggtgaagaa tctccaattga acaagcaaagaaagtgatgactatgtttgtccaacgacaggtaagtttcagattaaccttgagcttgtgacacacac.

[0092] (42) Nucleotide sequence of LDHA-paired Cas9-sgRNA target site on mouse chromosome 4 and location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) ccactgagccatctatctctccaccccaattttgtttttcttaagctttgttaatcttcacactataaacagaaggctaagatgaa agcatcaccaagtgcaggcaA GG ttgttgccagtggcttcttagactctgccctttg taatatgctaacaccttggtGGG ggcagaaggtggtacctcctccttggaaaatggcttacagctgaaaactgccacatgggtgttct.

[0093] (43) Nucleotide sequence of the paired Cas9-sgRNA target site on mouse chromosome 2 MCM8 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with a capital letter) aggagtgctcttgacttgtggaagacatgagtggtgcttat agaggcagaggttttggacgAGG aagattccaaagctggaaaagaggaagaggtgg tgggaacttc tcaggaaggTGG agagaaagagaaaacagagttgacctgaatgaagcttcaggaaagcacgcttctggtacgtgcacgcagggatcgatatgttttctctcgggaaagagacaattcaagggccggtagatgccagagtag.

[0094] (44) Nucleotide sequence of the Cas9-sgRNA target site at the first locus on MDC1 of mouse chromosome 17 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) cacttgggaagcagaggcaaggggatctctgagtttgaggccagcctggtctgattatgtgtttcctttacagatcatggaaagca CCCaggtgat tgactgggatgct gaagaagaggaagagacagaactatccagtggctccttggggtatagtgtggag CCTataggg cagctacgtctctt cagtggaactcatggaccagaaagaggtcaggagatattgggaactaaagtatgattgtgggtctttcttagaagaagttggaagttggaggg.

[0095] (45) Nucleotide sequence of the Cas9-sgRNA target site at the second site on MDC1 of mouse chromosome 17 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) ggaaatcatcacctcatggttgttcacatctttaatcccagcacttgggaagcagaggcaaggggatctctgagtttgaggccagcctggtctgattatgtgtttcctttacagatcatggaaagca CCCaggtgattgactgggatgct gaagaagaggaagagacag aac tatccagtggctccttGGG gtatagtgtggagcctatagggcagctacgtctcttcagtggaactcatggaccagaaagaggtcaggagatattgggaactaaagtatgattgtgggtctttcttagaagaagttggaagttggagggttggtagaaccatttat.

[0096] (46) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd site on MDC1 of mouse chromosome 17 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) cacttgggaagcagaggcaaggggatctctgagtttgaggccagcctggtctgattatgtgtttcctttacagatcatggaaagca CCCaggtgat tgactgggatgct gaagaagaggaagagacagaactatccag tggctccttggggtatagtgTGG agcctatagggcagctacgtctcttcagtggaactcatggaccagaaagaggtcaggagatattgggaactaaagtatgattgtgggtctttcttagaagaagttggaagttggaggg.

[0097] (47) Nucleotide sequence of the Cas9-sgRNA target site at the 4th site on MDC1 of mouse chromosome 17 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) cacttgggaagcagaggcaaggggatctctgagtttgaggccagcctggtctgattatgtgtttccttt acagatcatggaaagcacccAGG tgattgactgggatgctgaagaagaggaagagacagaactatccagtggctccttggggtatagtgtggag CCTataggg cagctacgtctctt cagtggaactcatggaccagaaagaggtcaggagatattgggaactaaagtatgattgtgggtctttcttagaagaagttggaagttggaggg.

[0098] (48) Nucleotide sequence of the paired Cas9-sgRNA target site at the 5th locus on MDC1 of mouse chromosome 17 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) cacttgggaagcagaggcaaggggatctctgagtttgaggccagcctggtctgattatgtgtttccttt acagatcatggaaagcacccAGG tgattgactgggatgctgaagaagaggaagagacagaactatccag tggctccttggggtatagtgTGG agcctatagggcagctacgtctcttcagtggaactcatggaccagaaagaggtcaggagatattgggaactaaagtatgattgtgggtctttcttagaagaagttggaagttggaggg.

[0099] (49) Nucleotide sequence of the Cas9-sgRNA target site at the first locus on NBS1 of mouse chromosome 4 and the location of the sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) gagaatgtgctcagaaccctcactgcagctggcgcttcgctttttaaagtgcttcctcccatttccagcacaagaaactcagctcggcagttgctttcggaggtggagaag CCAggctgatggcagaagacgac gaagaggaacagagcttcttttcagctcccggaa CCTg cgttgttgatgtaggaata acgaatacacagctcataatttcacactcccagaaaaaatggattcatttgataatggatacacttcaaaggtacataattctttcttcacaaattaaaaatgcaagtagcttttacatgcaactcagttaatacccaaagagtctacaagggcttgtca.

[0100] (50) Nucleotide sequence of the Cas9-sgRNA target site at the second locus on NBS1 of mouse chromosome 4 (underlined; -NGG PAM or its complementary sequence is marked with capital letters) gtcctgtatcatgaatgcgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtaccttacattcatttacttctgtttccctcaggagaaccataccgacttttgg ccggcgtggagtacgttgtTGG gaggaaaaactgtggcattctgattgaaaatgatcagtcaatcagtcgaaa CCAtgctgtcttaacagtaaact tccctgtaaccagtttggtatgctgctaactttattttactatctctttttgtttgctttaagatttatttatttattttatgcatatgagtgttgtatctgcatggacacc.

[0101] (51) Nucleotide sequence of the target Cas9-sgRNA paired with PIF1 on mouse chromosome 9 and location of the target sgRNA (underlined; -NGG PAM or its complementary sequence is marked with capital letters) caccggagatgccttctagcacagaggcggcgaccgatgaatgtgacgatgcggagctccggtgccgggtagccgtggaggagctgagt CCTggagggcaacctcgc aagc gccaggccctgcgcg CCGcagagctgagcctaggtcga aacgaacgacgtgagttaatgctgcgactgcaggcaccgttt.

[0102] (52) Nucleotide sequence of the Cas9-sgRNA target site at the first site on the POLQ of mouse chromosome 16 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) actgtgtcgtgtcctgaacagtcgagaggagcttgtttgcaatgagtcttccgcgccggagtaggaaacggcggcgttcatcgtccggct CCGaca cgttctcgggagatggt gatagctttgttagccctcagctccggtgtgga CCGgtgctgagtccacctcc ggggctgggacgcggccggaggctcacagggacaggtactcgacccgcggggccgacgcggccgagggcagcaggggttgagatgggctgcgagagt.

[0103] (53) Nucleotide sequence of the Cas9-sgRNA target site at the first locus on Rosa26 of mouse chromosome 6 and the location of the sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgggcgaaaaatgagttgctggtgaagacgttacacaagtaacatgagaaagcagaaaatgcaggt catccacgcacccctgacccAGG ccagcagggcggg ctgcagcatcagtacacAGG agaaagatccttattcctaagaatgagaaaggcaaaggcgcccgatagaataaattagcatagaaggggctttcccaggagttaaaactttccttctgagcgattacctactaaaaccag.

[0104] (54) Nucleotide sequence of the Cas9-sgRNA target site at the second locus on Rosa26 of mouse chromosome 6 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) Aaagagcccagtacttcatatccatttctcccgctccttctgcagccttatcaaaaggtattttagaacactcattttagccccattttcatttattatactggctta tccaacccctagacagagcatTGG………..agcactctggaggcagagacAGG cagatctctgagtttgagcccagcctggtctacacatcaagttctatctaggatagccaggaatacacacagaaaccctgttggggaggggggctctgagatttcataaaattataattgaagcattccctaatgagcc.

[0105] (55) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd locus on Rosa26 of mouse chromosome 6 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) atgagttgctggtgaagacgttacacaagtaacatgagaaagcagaaaatgcaggtcatccacgcacccctgacccagg CCAgcagggc ggg ct gcagcatc agtacacaggagaaagatccttatt cctaagaatgagaaaggcaaAGG cgcccgatagaataaattagcatagaaggggctttcccaggagttaaaactttccttctgagcgattacctactaaaaccagggcttttgcccactaccatttacctaggatcttggcttgcacggattc.

[0106] (56) Nucleotide sequence of the first site-paired Cas9-sgRNA target on a single copy of the sGEJ reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcga cctgcagcccaagc tctagcCGG actcagatctcgagctcaagcttcgaattcattaccctgttatccctaaccgccgccacca tggatt accctgttatccctAGG atccatccttcacatgatcagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaag ttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccacccttacgtacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggag.

[0107] (57) Nucleotide sequence of the second site paired with Cas9-sgRNA target on a single copy of the sGEJ reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcga cctgcagcccaagc tctagcCGG actcagatctcgagctcaagcttcgaattcattaccctgttatccctaaccgccgccaccatggattaccctgttat CCCtaggatccatccttcacat gatcagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatct gcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccacccttacgtacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggag.

[0108] (58) Nucleotide sequence of the 3rd site paired with Cas9-sgRNA target on a single copy of the sGEJ reporter system in mouse embryonic stem cells and the location of the paired sgRNA target (underlined; -NGG PAM or its complementary sequence is marked with capital letters) caccgcatctccgggcctttcgacctgcagcccaagctctag CCGgactcagatctcgagctcaa gcttcgaattcattaccctgttatccctaaccgccgccacca tggattaccctgttatccctAGG atccatccttcacatgatcagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgttt.

[0109] (59) Nucleotide sequence of the paired Cas9-sgRNA target site at the first locus on mouse chromosome 6 Rad52 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgggaagagatgctggatctgggagctcagagacggcttcactgtgcaaaaatggcatacataccaatttgagttccttctgcattatagagccagtatacagcggatgaataccagg CCA tccagaaagctctgagacagagagactgggtccagagtacattagcagccgcatggc TGG aggaggtcagaaggtaagcctccggagtcgtgggcatgtatctagttgttgacagaagatcctgc.

[0110] (60) Nucleotide sequence of the paired Cas9-sgRNA target site at the second locus on mouse chromosome 6 Rad52 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) tgggaagagatgctggatctgggagctcagagacggcttcactgtgcaaaaatggcatacataccaatttgagttccttctgcattatagagccagtatacagcggatgaataccagg CCA tccagaaagctctgagacagagagactgggtccagagtacattagcagccgcatggctgg AGG aggtcagaaggtaagcctccggagtcgtgggcatgtatctagttgttgacagaagatcctgc.

[0111] (61) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd locus on mouse chromosome 6 Rad52 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgggaagagatgctggatctgggagctcagagacggcttcactgtgcaaaaatggcatacataccaatttgagttccttctgcattatagagccagtatacagcggatgaataccagg CCA tccagaaagctctgagacagagactgggtccagagtacattagcagccgcatggctggagg AGG tcagaaggtaagcctccggagtcgtgggcatgtatctagttgttgacagaagatcctgc.

[0112] (62) Nucleotide sequence of the Cas9-sgRNA target site at the second site on the POLQ of mouse chromosome 16 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) actgtgtcgtgtcctgaacagtcgagaggagcttgtttgcaatgagtcttccgcgccggagtaggaaacggcggcgttcatcgtccggct CCG acacgttctcgggagatggtgatagctttgttagccctcagctccggtgtggac CGG tgctgagtccacctccggggctgggacgcggccggaggctcacagggacaggtactcgacccgcggggccgacgcggccgagggcagcaggggttgagatgggctgcgagagt.

[0113] (63) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd site on the POLQ of mouse chromosome 16 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) actgtgtcgtgtcctgaacagtcgagaggagcttgtttgcaatgagtcttccgcgccggagtaggaaacggcggcgttcatcgtccggct CCG acacgttctcgggagatggtgatagctttgttagccctcagct CCG gtgtggaccggtgctgagtccacctccggggctgggacgcggccggaggctcacagggacaggtactcgacccgcggggccgacgcggccgagggcagcaggggttgagatgggctgcgagagt.

[0114] (64) Nucleotide sequence of the paired Cas9-sgRNA target site at the first locus on human chromosome 19 AAVS1 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgggaccaccttatattcccagggccggttaatgtggctctggttctgggtacttttatc tgtcccctccaccccacagtGGG gccactagggacaggattggtgacagaaaagccccatccttaggcctcctcctt CCTagtctcctgatattgggtct aacccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctccttgccagaacctctaaggtttgcttacgatg.

[0115] (65) Nucleotide sequence of the Cas9-sgRNA target site at the second locus on human chromosome 19 AAVS1 and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) tgggaccaccttatattcccagggccggttaatgtggctctggttctgggtacttttatctgtcccctccaccccacagtg gggccactaggga caggatTGG tgacagaaaagccccatccttaggcctcctcctt CCTagtctcctgatattgggtct aacccccacctcctgttaggcagattccttatctggtgacacacccccatttcctggagccatctctctccttgccagaacctctaaggtttgcttacgatg.

[0116] (66) Nucleotide sequence of the paired Cas9-sgRNA target site at the first locus on human chromosome 11 (HBB) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) gggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacccaagagtcttctctgtctccacatgcccagtttctattggtctccttaaa CCTgtcttgtaaccttgatacca acctgcccagggcctcaccaccaacttcatc cacgttcaccttgcccc aca GGG cagtaacggcagacttctcctcaggagtcagatgcaccatggtgtctgtttgaggttgctagtgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctg.

[0117] (67) Nucleotide sequence of the Cas9-sgRNA target site at the second locus on human chromosome 11 (HBB) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) gggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacccaagagtcttctctgtctccacatgcccagtttctattggtctccttaaa CCTgtcttgtaaccttgatacca acctgcccagggcctcaccaccaacttcatccacgttcaccttgccccacagggcagtaacggcagacttct CCTcaggagtcagatgcaccatg gtgtctgtttgaggttgctagtgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctt.

[0118] (68) Nucleotide sequence of the Cas9-sgRNA target site at the 3rd locus on human chromosome 11 (HBB) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) gggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacccaagagtcttctctgtctccacatgcccagtttctattggtctccttaaa CCTgtcttgtaaccttgatacca acctgcccagggcctcaccaccaacttcatccacgttcaccttgccccacagggcag taacggcagacttctcctcAGG agtcagatgcaccatggtgtctgtttgaggttgctagtgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctg.

[0119] (69) Nucleotide sequence of the Cas9-sgRNA target site at the 4th locus on human chromosome 11 (HBB) and the location of the sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) gggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacccaagagtcttctctgtct CCAcatgcccagtttctattggt ctccttaaacctgtcttgtaaccttgataccaacctgcccagggcctcaccaccaacttcatc cacgttcaccttgcccc acaGGG cagtaacggcagacttctcctcaggagtcagatgcaccatggtgtctgtttgaggttgctagtgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctg.

[0120] (70) Nucleotide sequence of the paired Cas9-sgRNA target site at the 5th locus on human chromosome 11 (HBB) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with capital letters) gggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacccaagagtcttctctgtct CCAcatgcccagtttctattggtctccttaaacctgtcttgtaaccttgataccaacctgcccagggcctcaccaccaacttcatccacgttcaccttgccccacagggcagtaacggcagacttct CCTcaggagtcagatgcaccat ggtgtctgtttgaggttgctagtgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctg.

[0121] (71) Nucleotide sequence of the paired Cas9-sgRNA target site at the 6th locus on human chromosome 11 (HBB) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) gggtgggaaaatagaccaataggcagagagagtcagtgcctatcagaaacccaagagtcttctctgtct CCAcatgcccagtttctattggt ctccttaaacctgtcttgtaaccttgataccaacctgcccagggcctcaccaccaacttcatccacgttcaccttgccccacagggcag taacggcagacttctcctcAGG agtcagatgcaccatggtgtctgtttgaggttgctagtgaacacagttgtgtcagaagcaaatgtaagcaatagatggctctg.

[0122] Construction of sgRNA expression plasmid: 1) sgRNA Sequence Design and Synthesis: After identifying the target PAM structure (5'-NGG-3') in the target gene sequence, design 19bp or 20bp sgRNAs. Synthesize them as single strands according to the following example and anneal them to form a double-stranded structure for later use. If the first base of the sgRNA is A, C, or T, then excluding the structure complementary to the BbsI cleavage terminus, the overall sgRNA length is 20bp, as shown in the following example (N represents the base composition of the target genomic DNA): 5'-CACCG NNNNNNNNNNNNNNNNNNNN-3'oligo1 3'-C NNNNNNNNNNNNNNNNNNNN CAAA-5'oligo2 If the first base of the sgRNA is G, then the total length of the sgRNA, excluding the structure complementary to the end of the BbsI cleavage, is 19 bp, as shown in the following example (N represents the base composition of the target genomic DNA): 5'-CACCG NNNNNNNNNNNNNNNNNNN-3'oligo1 3'-C NNNNNNNNNNNNNNNNNNN CAAA-5'oligo2 2) The px330-U6-gRNA plasmid was digested with BbsI enzyme. After DNA gel identification, the linear DNA was recovered from the gel for later use. The in vitro digestion system was as follows: 0.5 μg plasmid DNA, 1 μL BbsI restriction enzyme, 2 μL 10X buffer, and double-distilled water to a total volume of 20 μL.

[0123] Enzyme digestion overnight at 37°C.

[0124] 3) Ligate the annealing product from step 1) and the linear DNA from step 2) to form a recombinant plasmid. The recombinant plasmid ligation system is as follows: 20 nM linear DNA, insert fragment (i.e., annealing product). Incubate overnight at 4°C. Transform the ligation product, select single clones, and perform Sanger sequencing to confirm successful insertion of the sgRNA into the expression vector.

[0125] Cell line transfection experiment: Well-grown mouse embryonic stem cells were transfected via suspension transfection. The well plates needed to be pre-treated with gelatin. Solution A and solution B were thoroughly mixed within 5 minutes and incubated at room temperature for 20 minutes to obtain the transfection solution. The transfection solution (volume shown below) was then added to the cell culture dish, followed by 200 μL of cell suspension. After incubation at room temperature for 6 hours, mouse embryonic stem cell culture medium was added to a final volume of 1 mL. The cells were then incubated at 37°C for 24 hours, after which the medium was replaced with fresh medium. In this experiment, the cell-level editing efficiency of the sgRNA library was validated using 6-well plates, and the transfection protocol was as follows: NIH-3T3 cells and U2OS cells were transfected to adherent cells. The day before transfection, the cells were packed at a density of 1*102 5 The plate is laid up one well per well, and then transfection is performed the next day. The following is the protocol (taking one well of a 24-well plate as an example): Total DNA amount: 0.8 μg Solution A Opti-MEM 50μL+Lipofectamine 2000 1.5μL Solution B: Opti-MEM 50μL + DNA 0.8μg Mix solution A and solution B thoroughly within 5 minutes and incubate at room temperature for 20 minutes to obtain the transfection solution. Before transfection, replace the medium with fresh mouse embryonic stem cell culture medium (300 μL / well). Then, add 102.3 μL of transfection solution to the cell culture dish and incubate at 37°C for 24 hours before replacing with fresh medium. 2. Selection and design of the orientation and spacing of paired sgRNAs at the target site

[0126] Templated insertion reduces the efficiency of precise NHEJ, and we need to optimize the paired Cas9-sgRNA system to reduce the occurrence of templated insertion. According to previously reported work in the field, templated insertion is generated by Cas9-induced 5'-sticky end completion (…). Figure 4 We hypothesize that template insertion can be reduced by controlling the position of paired Cas9-sgRNAs. Since the position of paired Cas9-sgRNAs at the target site is determined by the PAM (Polymer Alpha-Acid Matrix), and these PAM structures at DNA double-stranded targets (such as the PAM 5'-NGG-3' corresponding to Streptococcus pyogenes SpCas9) can be on either the Watson strand (W) or the Crick strand (C), the arrangement of paired Cas9-sgRNAs at the target site can combine into four forms: W / W, W / C, C / W, and C / C. Here, the first letter represents the strand on which the PAM is located at the upstream target site, the second letter represents the strand on which the PAM is located at the downstream target site, and C and W indicate that the PAM is on the Crick and Watson strands, respectively.

[0127] The Cas9 protein can produce both blunt ends and 5' extensions. When two blunt ends ligate, this results in the expected precise NHEJ. However, if there is a 5' extension, it may be filled in by DNA polymerase during the repair process. In this case, based on analysis and inference, only the W / C combination does not produce additional template insertion, while the other three combinations will (…). Figure 4 ).

[0128] Data analysis of 71 targets validated this hypothesis. Figure 5 (a). Meanwhile, since W / C basically does not produce templated insertions, the efficiency of precise NHEJ is also higher than that of W / W, C / W, and C / C combinations ( Figure 5 (b) This result suggests that when using paired Cas9-sgRNA technology, it is advisable to select "W / C" PAM combinations whenever possible to obtain a higher proportion of precise NHEJ repair events.

[0129] Paired Cas9-sgRNA gene editing, especially precise deletion of specific genomic fragments, requires consideration of the spacing between paired Cas9-sgRNAs at the target sites. Paired Cas9-sgRNA-mediated deletion of long genomic fragments (greater than 1 kb) has been previously applied, such as deleting genes encoding lncRNAs and entire exons of certain genes. Precise NHEJ has also occurred in these applications, but the frequency is unclear. However, studies have shown that the longer the spacing between two DSBs, the worse the repair efficiency of Group I NHEJ and precise NHEJ. To fully utilize precise NHEJ in CRISPR / Cas9 gene editing, the spacing between paired Cas9-sgRNAs designed at the aforementioned 71 genomic sites ranged from 12 to 148 base pairs (except for the 54th site, which had a spacing greater than 1000 base pairs). Analysis of changes in the spacing at these 71 sites revealed no correlation with changes in precise NHEJ efficiency. Figure 6 (a) indicates that the spacing between paired Cas9-sgRNAs does not affect the efficiency of precise NHEJ. To eliminate the interference of PAM position combinations of paired Cas9-sgRNAs, we further analyzed sequencing data of target NHEJ changes under the same w / w combination, and found no significant effect of the spacing between paired Cas9-sgRNAs on the efficiency of precise NHEJ. Figure 6 (b). Moreover, this short spacing is suitable for operation within most exons, overcoming the exon length limitation. 3. Testing the efficiency of paired Cas9-sgRNA gene editing

[0130] Based on the editing objectives, and following the selection and design principles for the orientation and spacing of paired sgRNAs at the target site as described in section 2 above, paired sgRNAs were designed. The site shown is human AAVS1 (adeno-associated virus integration site 1). Figure 7 The loci shown in (a) and HBB (β-globin gene) are ( Figure 7Taking b) as an example, we first used the general sgRNA design method to design 5 sgRNAs each (ghAAVS1-1, ghAAVS1-2, ghAAVS1-3, ghAAVS1-4 and ghAAVS1-5 for AAVS1; ghHBB-1, ghHBB-2, ghHBB-3, ghHBB-4 and ghHBB-5 for HBB), and paired them into 6 pairs. The pairs for AAVS1 or HBB are 1-3 (i.e., ghAAVS1-1 paired with ghAAVS1-3 or ghHBB-1 paired with ghHBB-3, the same below), 1-4, 1-5, 2-3, 2-4 and 2-5, and then constructed the expression vectors of sgRNAs. To test the gene editing efficiency of paired sgRNAs (using human AAVS1 and HBB sites as examples), we used a common transfection method in conjunction with a Cas9 expression plasmid to transfect HEK293 cells (or target cells, or mouse embryonic stem cells representing mouse cells and HEK293 cells representing human cells). After 72-96 hours of transfection, genomic DNA was collected, the target sequence was amplified by PCR, and the PCR products were analyzed by DNA gel electrophoresis. The WT group, serving as a blank control, was transfected with the Cas9 expression plasmid and the empty vector of paired sgRNA; therefore, no editing should have occurred at the target site. The gene editing efficiency of paired Cas9-sgRNAs mainly depends on the cleavage efficiency of the paired Cas9-sgRNAs at the target site.

[0131] If the Cas9-sgRNA cleaves efficiently, the spacer sequence will be deleted, resulting in two distinct bands on the DNA electrophoresis gel: one band is mainly similar to the unedited WT, and the other band is mainly the DNA band with the spacer sequence deleted, which is smaller than the WT. Figure 7 (c) Clearly, the most effective AAVS1 paired sgRNAs are ghAAVS1 / ghAAVS4 (W / C, 66bp) and ghAAVS2 / ghAAVS4 (W / C, 46bp), followed by ghAAVS1 / ghAAVS5 (W / W, 113bp) and ghAAVS2 / ghAAVS5 (W / W, 93bp), while ghAAVS1 / ghAAVS3 (W / W, 74bp) and ghAAVS2 / ghAAVS3 (W / W, 54bp) are essentially ineffective. HBB paired sgRNAs are generally effective, but there are no W / C combinations. We can select a pair of effective paired sgRNAs as needed for subsequent target gene editing. For example, we can choose ghAAVS1 / ghAAVS4 for full-frame deletion of endogenous AAVS1, and choose ghAAVS2 / ghAAVS4 for a 3n+1-bp frameshift mutation to delete AAVS1. 4. Improved gene knockout efficiency and homogeneity of precise deletion

[0132] To clarify whether paired Cas9-sgRNA technology is more effective than commonly used CRISPR / Cas9 protocols in gene knockout, we reanalyzed the deep sequencing data of four NHEJ products (i.e., Group I, II, III, and IV products) generated by paired Cas9-sgRNA at the aforementioned 71 genomic loci using our developed bioinformatics analysis software (Feng et al., Nucleic Acids Research, 2017, 45: 10614-10633), comparing the impact of these products on frameshift mutation efficiency. The paired Cas9-sgRNA strategy can be divided into idealized paired Cas9-sgRNA technology ("Ideal") and conventional gene editing technology ("Common"). The ideal "Ideal" technology only produces Group I repair, while the "Common" conventional strategy does not produce Group I events, only the sum of Group II, III, and IV NHEJ events. Figure 8 (a) Using paired Cas9-sgRNA technology (i.e., the "Paired" strategy), by setting the length of the paired Cas9-sgRNA spacer sequence, the precise NHEJ can precisely delete 3n (full frame), 3n+1 (frameshift), and 3n+2 (frameshift) base pairs. Figure 8 In the diagram, 'a' represents an integer 0 or greater than 0. Of the 71 genomic loci previously identified, 50 were designed as frameshift mutations (3n+1 or 3n+2 frameshifts). Therefore, we analyzed the deep sequencing results of these 50 loci, classifying the NHEJ products at each locus using the "Paired" strategy, the "Ideal" strategy, and the "Common" strategy, and calculating the proportion of 3n+1 and 3n+2 products in each category.

[0133] We found that, compared to the traditional "Common" strategy, once the frequency of precise NHEJs exceeds 29.8%, the "Paired" strategy can improve gene knockout (frameshift mutation) efficiency. The higher the frequency of precise NHEJs, the higher the frameshift mutation frequency increases, but it is lower than that of the "Ideal" strategy. Figure 8 (b). However, due to the high frequency of +1nt templated insertions, the 3n+1 base pairs in the spacer sequence will be transformed into 3n base pairs of whole-frame deletions due to +1nt templated insertions, affecting gene knockout (frameshift) efficiency. In fact, when +1nt templated insertions exceed 20.5%, the 3n+1 base pairs in the spacer actually reduce the frameshift mutation efficiency (compared to the "Common" traditional strategy), while the impact of the 3n+2 base pairs in the spacer is not significant. Figure 8 (c)

[0134] Overall, a 3n+2 base pair interval improves the gene knockout efficiency of frameshift mutations, while a 3n+1 base pair interval is less effective. Figure 8 (d). Therefore, the spacer sequence is best designed as 3n+2 base pairs.

[0135] It is foreseeable that, because the overall precision NHEJ in the paired Cas9-sgRNA strategy can reach 50%, the number of gene knockout products with specific length deletions will increase, and the homogeneity of gene knockout will also improve. 5. Improved efficiency and homogeneity of endogenous gene specific segment whole-frame deletion (applications include precise deletion of gene segments encoding specific structural domains or specific functional motifs).

[0136] In CRISPR gene editing applications, there are times when it is necessary to delete a specific gene segment entirely without affecting the function of other gene segments. For example, to study the function of a specific domain, the entire coding region of that domain may need to be deleted. However, due to technological limitations, this application is very limited and difficult to achieve using traditional gene editing methods. However, because the paired Cas9-sgRNA strategy can effectively delete 3n base pairs, it can be used to delete specific gene segments entirely.

[0137] Of the 71 genomic loci tested above, 20 were designed for whole-frame deletion (3n). After transfecting cells with paired Cas9-sgRNA expression plasmids, editing the target sites, and performing deep sequencing of PCR amplicon sequences, we re-analyzed the deep sequencing results of these 20 whole-frame deletion mutation sites using the aforementioned bioinformatics software (Feng et al., Nucleic Acids Research, 2017, 45: 10614-10633) while analyzing 50 frameshift mutations. We categorized the NHEJ products at each site using the "Paired" strategy, the "Ideal" strategy, and the "Common" strategy, and calculated the proportion of 3n products.

[0138] Our data show that, compared to the traditional "Common" strategy, the "Paired" strategy can improve the efficiency of whole-frame genome deletion, increasing it by an average of 30% to approximately 50%. Figure 9 (a) The higher the frequency of the precise NHEJ, the higher the level of integer mutation improved by the "Paired" pairing strategy, up to a factor of 2, from about 33% to 66%, but overall lower than "Ideal" (a). Figure 9 (b) In particular, the "Common" traditional strategy involves deletions of varying lengths and exhibits high heterogeneity, while the "Paired" strategy, due to its precise NHEJ repair, effectively deletes DNA fragments of specific lengths, thus exhibiting high homogeneity.

[0139] In summary, the "W / C" type PAM combination can achieve a higher proportion of precise NHEJ repair events. For frameshift mutations, a 3n+1 or 3n+2 base pair interval between paired sgRNAs is sufficient; for whole-frame deletions, a 3n base pair interval between paired sgRNAs can significantly improve deletion efficiency. Example 2: Quantitative analysis of NHEJ based on paired Cas9-sgRNA technology at cellular and animal levels

[0140] Cas9-sgRNA technology can design paired sgRNAs (sgRNA target site intervals of 23-148 base pairs) for Cas9 in any selected chromosomal region, such as the ALDHA intron 5 site (AGCATCACCAAGTGCAGGCA and TAATATGCTAACACCTTGGT) and ROSA26 site (CATCCACGCACCCCTGACCC and GGGCTGCAGCATCAGTACAC) in mouse cells. The paired sgRNAs are combined with Cas9 expression plasmids and transfected into mouse embryonic stem cells. Three days later, the genomic DNA of the cells is collected, and NHEJ repair sites are amplified by PCR. The PCR amplification products are then subjected to deep sequencing, and NHEJs are quantitatively analyzed using the aforementioned bioinformatics analysis methods. Based on the analysis results, NHEJ repair maps of these two targets are constructed, including the total NHEJ efficiency (i.e., total editing efficiency), the frequency and ratio of precise NHEJs and mutant NHEJs, the direction, length and frequency of addition and deletion of mutant NHEJ interfaces, and the utilization of micro-homologous sequences of mutant NHEJ interfaces. To validate this NHEJ analysis method for the no-reporting system, we compared it with XRCC4 using this method. + / + Mouse embryonic stem cells and XRCC4 - / - NHEJ of mouse embryonic stem cells.

[0141] The results showed that, consistent with the known NHEJ function of XRCC4, XRCC4 deletion at both LDHA and ROSA26 sites led to a significant decrease in overall NHEJ efficiency. Figure 10 (a). The absence of XRCC4 also caused a decrease in the repair frequency of Group I NHEJ ( Figure 10 (b) This may be because the reduced efficiency of precise NHEJ leads to the cessation of the "re-cutting-re-repairing" cycle of paired Cas9-sgRNAs, while the probability of deleting the spacer sequence decreases. The proportion of mutant NHEJs in the remaining Group I NHEJs is significantly increased, while the proportion of precise NHEJs is low (b). Figure 10 (c) In the presence of XRCC4, the deletion frequency of 58-63 bp at the NHEJ interface of the LDHA site (30.05%) was significantly higher than that in the absence of XRCC4 (6.62%) (χ²).2 (test: P < 0.0001), but when the deletion length was greater than 63 bp, the frequency of occurrence in XRCC4 wild-type cells (69.95%) was lower than that in XRCC4-deficient cells (93.38%) (χ²). 2 test: P < 0.0001). At the ROSA26 site, the deletion frequency of 34-39 bp at the interface (41.45%) was significantly higher when XRCC4 was present than when XRCC4 was absent (11.31%) (χ²). 2 (test: P < 0.0001), but when the deletion length was greater than 39 bp, the frequency of occurrence in XRCC4 wild-type cells (58.55%) was lower than that in XRCC4-deficient cells (88.69%) (χ²). 2 test: P < 0.0001) Figure 11 (a). When XRCC4 is missing, the deletion length at the NHEJ interface is significantly increased ( Figure 11 (a) This suggests that XRCC4 drives precise NHEJ. Furthermore, XRCC4 - / - Increased utilization of microhomological sequences in mouse embryonic stem cells ( Figure 11 (b) Furthermore, XRCC4 deletion leads to a greater tendency for cells to utilize longer microhomologous sequence fragments. These results indicate that XRCC4 inhibits terminal processing in NHEJs and its dependence on microhomologous sequences, consistent with the known NHEJ function of XRCC4. Therefore, paired Cas9-sgRNA technology can be used as an alternative to reporter systems for the quantitative analysis of intracellular NHEJs.

[0142] Because paired Cas9-sgRNA technology is not limited by reporter systems and the genomic sites where reporter systems are integrated, it can be used for quantitative analysis of in vivo organ-specific NHEJ. To assess this possibility, we used hydrodynamic tail vein injection. The injection conditions were: 200 μg of Cas9 expression plasmid and 150 μg of paired sgRNA (paired sgRNA at the mouse LDHA site) expression plasmid dissolved in 1.8 mL of physiological saline. The injection time was controlled within 10 seconds. Paired Cas9-sgRNA expression plasmids targeting the LDHA intron 5 site were injected into the livers of 6-8 week old C57BL / 6 mice (Shanghai Nanmo). Figure 12 In step a), a plasmid expressing paired Cas9-sgRNA targeting the LDHA intron site was introduced into liver cells, inducing DSB at specific sites in mouse liver cells. Mice were sacrificed after 30 days, and two liver tissue samples were collected from each mouse. Genomic DNA was isolated and purified, and the NHEJ repair target was amplified by PCR. Next-generation sequencing and bioinformatics analysis were performed to assess NHEJ repair of the target DSB. The measured NHEJ included the total editing efficiency of the paired Cas9-sgRNA target (…). Figure 12(b) Distribution of Group I-IV in editing efficiency ( Figure 12 The frequencies of (c) precise NHEJ and mutant NHEJ (i.e., Del, Ins, and InDel) Figure 12 d), Deletion length and frequency of Group I mutant NHEJ interface ( Figure 13 (a) and the utilization of micro-homogeneous sequences in the Group I NHEJ interface ( Figure 13 (b) In mouse liver, the overall editing efficiency is approximately 1%-4%, which is much lower than the editing efficiency at the cellular level. Figure 12 (b) The frequency of Group I production was very similar across the four liver tissue samples, around 80%, while the proportion of precise NHEJ was around 50%. Figure 12 c and d in 12). In liver tissue, the base deletion patterns at the interface sites are very consistent ( Figure 13 (a), and the characteristics of micro-homologous sequences are almost identical ( Figure 13 (b). These results show that paired Cas9-sgRNA technology can be used to quantitatively analyze tissue- and organ-specific NHEJ in vivo. Example 3: Knockout of the MDC1 gene

[0143] To knock out the mouse MDC1 (gene ID: 240087) gene using paired Cas9-sgRNA technology, we designed three pairs of sgRNAs (mMDC1-1: ACAGATCATGGAAAGCACCC; mMDC1-2: AGCATCCCAGTCAATCACCT; mMDC1-3: AACTATCCAGTGGCTCCTT) in exon 2 of the MDC1 gene. Single sgRNAs and paired sgRNAs were transfected into mouse embryonic stem cells. Three days later, PCR amplification and DNA electrophoresis analysis revealed that the paired sgRNA2 / sgRNA3 (C / W, 52bp) had a higher editing efficiency. Figure 15 (a) Deep sequencing also showed that the efficiency of precise ligation was about 50%, and the templated insertion frequency was about 5%. Figure 15(b) Therefore, we selected this pair of sgRNAs to guide Cas9 MDC1 gene knockout and selected monoclonal cell lines from cells edited with paired Cas9-sgRNAs. Specifically, the selected paired Cas9-sgRNA expression plasmids were transfected into mouse embryonic stem cells. The transfected cells were diluted 24 hours later and seeded onto MEF (mouse embryonic fibroblast) feeder layers. Monoclonal cells formed after 5-6 days. Monoclonal cells were picked, digested, and seeded into 96-well plates pre-coated with MEF. After the cells proliferated to a sufficient quantity, they were passaged into 24-well plates. Cells from the confluent 24-well plates were collected, leaving a small portion for further culture. Genomic DNA was extracted from the collected cells and initial screening was performed by PCR. First-generation sequencing revealed that 18 out of 35 single clones had only one allele edited; two alleles in 8 clones were knocked out via precise NHEJ ligation; two alleles in 4 clones were knocked out, but at least one allele was knocked out via inaccurate NHEJ ligation; and 5 clones contained integer-length mutations. Figure 15 (c) This demonstrates that the paired Cas9-sgRNA editing strategy can efficiently produce homogeneous knockout products (i.e., 8 out of 35 clones were precisely knocked out homozygous). Western blotting confirmed that the MDC1 gene was indeed knocked out. Figure 15 (d).

[0144] The nucleotide sequence of the Cas9-sgRNA editing target site paired with the mouse chromosome 2 minor satellite (Misat) and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) cttaccatattcctcaaggtttccggggctagagaaagtttgctggtggtagacgaagccgactgcatggtagctgggtctcagttgtttgtggcct ccttagtagacagatggacaTGG tttgaattttttatattaagaaaaggattgggctacaaatagagctcagtcaatagactgcttgcccag CCTgtgtgcagccctgggtcaag catatgcatcccattatcctggcctcgtggctcttcttatagacccagcactgaataggtagaagctgtatattac.

[0145] The nucleotide sequence of the paired Cas9-sgRNA target site on mouse chromosome 3 TRPC21A and the location of the paired sgRNA target site (underlined; -NGG PAM or its complementary sequence is marked with uppercase letters) ccacagtgcagcacagttatagtaaagtggaacacttggacagcaaaatggagcactgtgacatcacaatggagcactgtgacaccatag tgcagcacagtggcagcac aGGG gactagtgtgacattacaat ggagcactgtgagaccttagTGG agcaaatgacaccacagtgagcactatgacacaaaatttgagccctgagacactttagtggagcactgtgatatcacattg. Example 4: Precise whole-code deletion of gene fragments encoding the 53BP1 domain

[0146] Paired sgRNA technology allows for controlled excision fragment length, enabling precise whole-frame excision. This allows for functional studies of specific gene fragments without frameshift mutations, particularly for studying fragments encoding lethal gene domains. To validate this application, we selected the 53BP1 gene (gene ID: 27223) as our research subject, focusing on the key domains Tudor and OD of 53BP1, which are essential for the recruitment of the 53BP1 protein to the DSB lesion site. Figure 16 (a) We designed paired sgRNAs for these two domains (Tudor domain: GGGAAGATCACCCGAGATGT and GGGTACGAATGTGACGTGCT; OD domain: GAGGTGTACGGACTTCTCGAA and TTATGTGGATGGGACAGAAG). The DNA fragments excised between these two pairs of paired sgRNAs are both 3n bases in length. This allows for precise ligation of the two ends using NHEJ during gene editing, achieving functional disruption of either the Tudor or OD domain without affecting other parts of 53BP1. Following the method described in Example 2, mouse embryonic stem cells were transfected with paired Cas9-sgRNAs. Single-clone cell lines with the Tudor or OD domains excised were selected, and first-generation sequencing confirmed that the selected clone gene sequences were precisely edited. Figure 16 (a). Western blotting analysis showed that although the Tudor or OD structure was removed, 53BP1 protein expression was not significantly affected. Figure 16(b) Using ionizing radiation to irradiate cells to generate DSBs, combined with immunofluorescence experiments, we found that wild-type 53BP1 cells could recruit to damaged sites to form focal points (foci) and co-localize with γH2AX focal points, while 53BP1 mutants lacking Tudor or OD could not form focal points. Figure 16 (c) This indicates that the 53BP1 protein lacking the Tudor or OD domain can no longer be recruited to the site of the DNA double-strand break, consistent with previous findings. The resulting 53BP1 mutant cells lacking either the Tudor or OD domain can be used for subsequent functional studies. Example 5: Measurement of cellular heterochromatin NHEJ using paired Cas9-sgRNA technology

[0147] Because DNA is packaged within chromatin structures, the generation and repair of DNA strand blight (DSB) are influenced by chromatin structure and histone modifications. Considering the differences in structure between euchromatin and heterochromatin, the repair and regulation of NHEJ in euchromatin and heterochromatin will differ. Therefore, in addition to analyzing euchromatin NHEJ... Figure 10 and Figure 11 We can also use paired Cas9-sgRNA technology to analyze heterochromatin NHEJ. We selected two murine constitutive heterochromatin genomic loci, Misat and TRPC21, and designed paired sgRNAs (Misat loci: CCTTAGTAGACAGATGGACA and CTTGACCCAGGGCTGCACAC; TRPC21 loci: TGCAGCACAGTGGCAGCACA and GGAGCACTGTGAGACCTTAG), combined with Cas9, and tested and analyzed the NHEJ at these loci (method as in Example 1), including the overall editing efficiency of specific targets (…). Figure 17 (a) Distribution of Groups I-IV in editing efficiency ( Figure 17 (b) The frequencies of precise NHEJ and mutant NHEJ (i.e., Del, Ins, and InDel) Figure 17 c), Deletion length and frequency of Group I mutant NHEJ interface ( Figure 18 (a) and the micro-homogeneous sequence utilization of the Group I mutant NHEJ interface ( Figure 18 (b) These results show that the paired Cas9-sgRNA technology can quantitatively analyze heterochromatin NHEJ in cells and organs, and study the molecular mechanism of heterochromatin NHEJ.

Claims

1. A method for designing paired sgRNAs for precise gene editing, wherein the paired sgRNA positions are in the form of W / C, where W represents PAM on the Watson strand and C represents PAM on the Crick strand.

2. In the method of claim 1, when the purpose of gene editing is to perform frameshift mutations or gene knockouts at target genes or target genomic sites, the spacing between paired sgRNAs is 3n+1 or 3n+2 base pairs; wherein, n is an integer.

3. In the method described in claim 1, when the gene editing objective is to perform full-frame mutations or precise full-frame deletions on the target gene or target genomic locus, the spacing between paired sgRNAs is 3n base pairs; wherein, n is an integer.

4. The application of the paired sgRNA designed by the method of claim 1 in improving the efficiency and accuracy of CRISPR / Cas9 gene editing.

5. A pairing sgRNA for gene editing, characterized in that... The paired sgRNAs are obtained as follows: the positions of the two PAMs corresponding to the paired sgRNAs on the target gene or genomic site are in the form of W / C; two sgRNAs with different spacings are selected as paired sgRNAs according to the gene editing requirements; when the gene editing is a whole-frame deletion, the cleavage spacing mediated by the paired sgRNAs is 3n base pairs; when the gene editing is a frameshift mutation or gene knockout, the cleavage spacing mediated by the paired sgRNAs is 3n+1 or 3n+2 base pairs. Where W represents the PAM on the Watson chain, C represents the PAM on the Crick chain, and n is an integer.

6. A method for pairing Cas9-sgRNA gene editing based on precise NHEJ repair, characterized in that, The method is as follows: (1) When the paired sgRNA performs frameshift mutation or gene knockout on the target gene or target genome site, the paired sgRNA position combination is W / C, and the spacing is 3n+1 or 3n+2 base pairs; (2) When the paired sgRNA performs whole-frame mutation or precise whole-frame deletion on the target gene or target genome site, the paired sgRNA position combination is W / C, and the spacing is 3n base pairs.

7. The method as described in claim 6, characterized in that, The method is operated according to the following steps: multiple sgRNAs are designed according to the target gene, and two sgRNAs with a spacing of 3n or non-3n base pairs are selected as paired sgRNAs according to the gene editing requirements. The position combination of paired sgRNAs is W / C. An sgRNA expression plasmid is constructed and introduced into cells together with the Cas9 expression plasmid for expression. The target DNA is amplified by PCR, and the working efficiency of the paired Cas9-sgRNA is detected by DNA electrophoresis. The high-efficiency paired sgRNA is selected for gene editing.

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