Endonuclease gs12-7max and gene editing system mediated thereby

Abstract

Disclosed in the present invention are a Gs12-7 endonuclease mutant and a gene editing system mediated thereby. When the amino acid at the 157the site of Gs12-7 endonuclease is mutated from glutamic acid (Glu) to arginine (Arg), the activity of the gene editing enzyme is significantly improved. The mutant is named Gs12-7MAX, and gene editing can be efficiently completed on the basis of a CRISPR-Gs12-7MAX system.

Description

Endonuclease Gs12-7MAX and gene editing system mediated by the same TECHNICAL FIELD

[0001] The present application belongs to the technical field of genome editing, and particularly relates to an endonuclease Gs12-7MAX and a gene editing system mediated by the same. BACKGROUND

[0002] The CRISPR / Cas system is widely used for genome site-directed modification, epigenetic, base and lead editing of human cells and animals and plants. The commonly used editing system is derived from the CRISPR / Cas9 of Streptococcus pyogenes, in addition to which, Cas12a nucleic acid endonuclease, including AsCas12a from Acidaminococcus sp. and LbCas12a of Lachnospiraceae bacterium ND2006, is also commonly used for genome editing and the like. The Cas12a nucleic acid endonuclease has the following advantages: first, it recognizes a PAM target site rich in T, which expands the genome editing targeting space; only about 40 nucleotides (nt) of CRISPR RNA (crRNA) are needed for specific editing of the target; it has RNase activity, and multiple targeting editing can be achieved through poly-crRNA transcript processing; in addition, studies have shown that Cas12a is more specific than SpCas9, and has lower off-target effects.

[0003] Although the Cas12a nuclease has shown various advantages, it is still limited by low editing efficiency at present, and therefore it is urgent to develop a high-efficiency Cas12a-based genome editing technology. Based on this, the present application further mutates the Gs12-7 endonuclease (CN116144631A) identified in the previous stage through bioinformatics and experiments, and develops Gs12-7MAX and a genome editing technology mediated by the same. SUMMARY

[0004] The present application first develops an endonuclease Gs12-7MAX, which has higher gene editing activity compared with wild-type Gs12-7 after mutating the 157th amino acid of Gs12-7 from Glu to Arg, and establishes a high-efficiency genome editing technology based on the CRISPR-Gs12-7MAX system.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0006] An endonuclease Gs12-7MAX comprises the following proteins:

[0007] I. Gs12-7MAX protein of the amino acid sequence shown in SEQ ID NO. 1, wherein the amino acid at position 157 is mutated from Glu to Arg, relative to the wild-type Gs12-7 endonuclease.

[0008] II. Protein having one or more substitutions, deletions, or additions of amino acids compared to the amino acid sequence shown in SEQ ID NO. 1, and substantially retaining the biological function derived from the sequence.

[0009] Fusion protein comprising the above-mentioned endonuclease and a polypeptide connected to the N-terminus or C-terminus of the protein.

[0010] Polynucleotide, which is a polynucleotide encoding the above-mentioned endonuclease, or a polynucleotide encoding the above-mentioned fusion protein. Vector or host cell containing the polynucleotide.

[0011] Application of the above-mentioned endonuclease in gene editing, including modification of prokaryotic genome, eukaryotic genome, or in vitro gene, knocking out genes, changing expression of gene products, repairing mutations, or inserting polynucleotides.

[0012] CRISPR / Gs12-7MAX gene editing system comprising the above-mentioned endonuclease, or fusion protein, or polynucleotide, or vector, or host cell. Further, it also comprises direct repeat sequence capable of binding to the above-mentioned endonuclease and crRNA capable of targeting target sequence.

[0013] The technical solutions of the present application have the following main beneficial effects:

[0014] 1. The present application first provides a Gs12-7MAX with high gene editing activity, wherein the amino acid at position 157 is mutated from Glu to Arg, relative to the wild-type Gs12-7.

[0015] 2. The present application develops a CRISPR / Gs12-7MAX system, which has the same level of editing activity in cells as the AsCas12a Ultra reported in the prior art, and its off-target effect on the whole genome level is significantly lower than that of AsCas12a Ultra.

[0016] 3. Based on the CRISPR-Gs12-7MAX system, the preparation of gene editing animal models can be efficiently completed. Given the characteristics of Gs12-7MAX, such as high editing activity, low off-target effect, and high model preparation capacity, it has broad application prospects in the field of genome editing. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1. Comparison of the genome editing activity of Gs12-7 and enAsCas12a-HFl based on high-throughput sequencing strategy. A. Comparison of the genome editing activity of Gs12-7 and enAsCas12a-HFl for single site by RNP delivery strategy; B. Analysis and statistical comparison of the genome editing activity of Gs12-7 and enAsCas12a-HFl based on plasmid delivery strategy.

[0018] Figure 2. Evaluation of the genome editing activity of two Gs12-7 variants, Gs12-7E157R (amino acid at position 157 mutated from Glu to Arg) and Gs12-7EGK (amino acid at position 157 mutated from Glu to Arg, amino acid at position 516 mutated from Gly to Arg and amino acid at position 522 mutated from Lys to Val), for single site based on high-throughput sequencing strategy. A. Diagram of the pattern of amino acid mutation sites of the two Gs12-7 variants; B. Comparison of the genome editing activity of Gs12-7E157R (Gs12-7MAX), Gs12-7EGK and enAsCas12a-HFl for single site; C. Statistical analysis and comparison of the genome editing activity of Gs12-7E157R (Gs12-7MAX), Gs12-7EGK and enAsCas12a-HFl.

[0019] Figure 3. Comparison of the genome editing activity of Gs12-7MAX, Gs12-7EGK and enAsCas12a-HFl for multiple different sites simultaneously based on high-throughput sequencing strategy. A. Comparison of the genome editing activity of Gs12-7MAX, Gs12-7EGK and enAsCas12a-HFl for multiple different sites simultaneously; B. Statistical analysis and comparison of the genome editing activity of Gs12-7MAX, Gs12-7EGK and enAsCas12a-HFl for multiple sites simultaneously.

[0020] Figure 4. Detection of the distribution of genome editing Indels of Gs12-7MAX, Gs12-7EGK and enAsCas12a-HFl based on high-throughput sequencing strategy. A. Distribution of genome editing Indels of Gs12-7MAX in HEK293T cells; B. Distribution of genome editing Indels of Gs12-7EGK in HEK293T cells; C. Distribution of genome editing Indels of enAsCas12a-HFl in HEK293T cells.

[0021] Figure 5. Assessing the specificity of Gs12-7MAX and enAsCas12a-HFl based on high-throughput sequencing strategy. A. Experimental detection of predicted off-target sites of Gs12-7MAX and enAsCas12a-HFl; B. Statistical analysis and comparison of actual occurrence of Gs12-7MAX and enAsCas12a-HFl against predicted off-target sites.

[0022] Figure 6. Assessing the editing activity and genome-wide specificity of Gs12-7MAX and AsCas12a Ultra based on high-throughput sequencing and GUIDE-seq. A. Comparison of editing activity of Gs12-7MAX and AsCas12a Ultra in HEK293T cells by high-throughput sequencing; B. Statistical analysis and comparison of editing activity of Gs12-7MAX and AsCas12a Ultra in HEK293T cells; C. Editing activity of four selected sites by GUIDE-seq; D. Insertion rate of dsODN of four selected target sites by GUIDE-seq; E. Genome-wide assessment of specificity of Gs12-7MAX and AsCas12a Ultra by GUIDE-seq.

[0023] Figure 7. Assessing the gene editing activity of Gs12-7MAX in mouse individual. A. Testing the gene editing activity of Gs12-7MAX in mouse by microinjection of Gs12-7MAX mRNA and crRNA targeting Tyr gene; B. Editing activity against two sites of Tyr gene in mouse embryo; C. Editing activity against two sites of Tyr gene in F0 generation mouse; D. Coat color phenotype of F0 generation mouse edited by Gs12-7MAX against two sites of Tyr gene, respectively. DETAILED DESCRIPTION

[0024] The application will be further described with reference to the following examples. It should be appreciated that these examples are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise indicated, the experimental procedures in the following examples were conducted in accordance with conventional procedures or as recommended by the manufacturer.

[0025] Example 1. Assessing the genome editing activity of endonuclease Gs12-7 and enAsCas12a-HFl

[0026] This example is directed to the endonuclease Gs12-7 (CN116144631A) previously developed by the inventors, and the activity comparison evaluation is carried out with the enAsCas12a-HF1 endonuclease with relatively high activity known at present. The expression vectors of the two endonucleases are respectively co-transfected into HEK 293T cells with the corresponding crRNA expression vectors, and the crRNA paired with the target nucleic acid is used to guide the Gs12-7 or enAsCas12a nuclease to recognize and bind on the target gene, so as to trigger the genome cleavage activity. First, the cells are collected and the genomic DNA is extracted, and then high-throughput sequencing is carried out, and then the CRISPResso2 software (https: / / github.com / pinellolab / CRISPResso2 / releases) is used to analyze the genome editing activity of the target site.

[0027] The target genes selected in this example are human DNMT1, EMX1, FANCF and RUNX1 genes, the PAM recognized by the crRNA is TTTV, and the corresponding crRNA sequences are shown in Table 1, and the underlined region is the target pairing region. The crRNA in Table 1 is constructed into a crRNA eukaryotic expression vector.

[0028] Table 1. Name and corresponding sequence of crRNA

[0029] Firstly, the activity of Gs12-7 is compared with the known enAsCas12a-HF1 endonuclease by RNP delivery strategy. When the confluence of HEK 293T cells reaches 70-80%, the cells are plated, and the number of cells inoculated in a 12-well plate is 8x10 4 cells / well, and the cells are plated for 6-8h before transfection. After incubation of 1.25μg Gs12-7 or enAsCas12a-HF1 protein and 625ng crRNA, the RNP complex of Gs12-7 or enAsCas12a-HF1 is formed, and is respectively mixed with 50μL opti-MEM and 2.6μL Cas9 plus TM reagent to form solution one; 3μL of CRISPR TMreagent, mix well to make solution two. Add solution two to solution one and mix well, incubate at room temperature for 10 min. Add the mixed solution to the culture medium with HEK 293T cells for transfection. After 72 h of culture at 37 °C, discard the culture medium, resuspend the cells with 100 μL PBS, and extract the genomic DNA of the cells. Perform PCR amplification on the target site of the positive transfection cells. Observe the changes in the cleavage target band by T7EN1 enzyme digestion and agarose gel electrophoresis to determine the genomic editing activity of Gs12-7 and enAsCas12a-HF1 endonuclease. Further calculate the gene editing efficiency by Image J. The template for the negative control is the genome of the wild-type HEK 293T cells without any treatment.

[0030] Secondly, the HEK 293T cells were plated when the confluence reached 70-80%, and the number of cells seeded in a 12-well plate was 8 x 10 4 cells / well. The cells were plated for 6-8 h for transfection. 1 μg of Gs12-7 eukaryotic expression vector plenti-Gs12-7-puro or enAsCas12a-HF1 eukaryotic expression vector plenti-enAsCas12a-HF1-puro, 500 ng of different crRNA expression vector plenti-U6-crRNA-zsGreen, and 10 μL of Jetprime regent were sequentially added to 200 μL of Jetprime Buffer, mixed well, and incubated at room temperature for 10 min. The mixed solution was added to the culture medium with HEK 293T cells for transfection. After 72 h of culture at 37 °C, the culture medium was discarded, the cells were resuspended with 100 μL of PBS, and the genomic DNA of the cells was extracted. The target site of the positive transfection cells was subjected to PCR amplification to edit the sequence nearby. The PCR product was recovered for amplicon sequencing by Illumina Novaseq PE150, and the editing activity was analyzed by CRISPResso2.

[0031] As shown in FIG. 1A, using RNP delivery cell genome editing experiments, it was found that both Gs12-7 and enAsCas12a-HF1 nucleases had high editing activity, and without positive gene editing cell enrichment, the gene editing activity of Gs12-7 was about 30%, but relatively, the editing activity of Gs12-7 nuclease was lower than that of enAsCas12a-HF1. In the plasmid delivery cell genome editing experiment, it was found that the average gene editing efficiency of Gs12-7 was 17.83%, while that of enAsCas12a-HF1 was 21.32%, and it was found that the average gene editing efficiency of wild-type Gs12-7 for multiple sites was still significantly lower than that of enAsCas12a-HF1 nuclease (FIG. 1B). In view of this, the amino acid sequence of the Gs12-7 endonuclease is further mutated to improve its genome editing activity.

[0032] Example 2. Comparison of genome editing activity of Gs12-7 different variants and enAsCas12a-HF1 in HEK293T cells

[0033] In this example, two variants of Gs12-7 were prepared (FIG. 2A): (1) variant Gs12-7E157R, in which the 157th amino acid is mutated from Glu to Arg, and the amino acid sequence is shown as SEQ ID NO: 1; (2) Gs12-7EGK, in which the 157th amino acid is mutated from Glu to Arg, the 516th amino acid is mutated from Gly to Arg, and the 522nd amino acid is mutated from Lys to Val, and the amino acid sequence is shown as SEQ ID NO: 3, and the genome editing ability of each at the cell level was evaluated with known enAsCas12a-HF1 endonuclease.

[0034] In this embodiment, the Gs12-7E157R human codon-optimized expression vector plenti-Gs12-7E157R-puro, the Gs12-7EGK human codon-optimized expression vector plenti-Gs12-7EGK-puro, and the enAsCas12a-HF1 expression vector are co-transfected into HEK 293T cells with the crRNA expression vector plenti-U6-crRNA-zsGreen, respectively, to guide the Gs12-7E157R, Gs12-7EGK, and enAsCas12a-HF1 proteins to recognize and bind to the target nucleic acid on the target nucleic acid matched with the crRNA, thereby triggering the genome cleavage activity. Finally, the cells are collected and the genomic DNA is extracted, and the editing activity of the target site is analyzed by high-throughput sequencing and using CRISPResso2. The nucleotide sequence of the Gs12-7E157R human codon-optimized expression vector is shown in SEQ ID NO. 2, and the nucleotide sequence of the Gs12-7EGK human codon-optimized expression vector is shown in SEQ ID NO. 4.

[0035] In this embodiment, the target nucleic acid is selected as the human EMX1, FANCF, and RUNX1 genes, and the PAM is TTTV. The design of the corresponding crRNA is shown in Table 2, and the underlined region is the targeting region.

[0036] Table 2. Name and corresponding sequence of crRNA

[0037] In this embodiment, the target nucleic acid is selected as the human EMX1, FANCF, and RUNX1 genes, and the PAM is TTTV. The design of the corresponding crRNA is shown in Table 2, and the underlined region is the targeting region. 4 In this embodiment, the target nucleic acid is selected as the human EMX1, FANCF, and RUNX1 genes, and the PAM is TTTV. The design of the corresponding crRNA is shown in Table 2, and the underlined region is the targeting region.

[0038] The results are shown in Figure 2. Both Gs12-7E157R and Gs12-7EGK have genome editing activity. Without enrichment of positive gene editing cells, Gs12-7E157R can reach an editing activity of about 42%, while the gene editing activity of Gs12-7EGK is lower, only about 28% (Figure 2B). Further detection found that for different editing sites, the gene editing activity of Gs12-7E157R is higher than that of enAsCas12a-HF1 (Figure 2B). Statistical analysis found that the average editing efficiency of Gs12-7E157R can reach about 30%, which is significantly higher than the currently known enhanced enAsCas12a-HF1 (Figure 2C). By comparison, the genome editing activity of Gs12-7E157R is relatively high, so it is named Gs12-7MAX.

[0039] Example 3. Comparison of Gs12-7MAX, Gs12-7EGK and enAsCas12a-HF1 simultaneous cleavage of multiple sites of gene editing activity

[0040] In this example, the activity of Gs12-7MAX, Gs12-7EGK and enAsCas12a-HF1 in simultaneously editing multiple different genomic sites in cells was evaluated. Gs12-7MAX, Gs12-7EGK and enAsCas12a-HF1 were co-transfected with crRNA eukaryotic expression vectors that can simultaneously target 3 or 2 genes, respectively. The crRNA paired with the target nucleic acid was used to guide Gs12-7MAX, Gs12-7EGK and enAsCas12a-HF1 protein to recognize and bind to the target nucleic acid, thereby triggering genome cleavage activity. Finally, HEK 293T cells were collected and genomic DNA was extracted, and the editing activity of the target site was analyzed by high-throughput sequencing and using CRISPResso2.

[0041] In this example, the target nucleic acid was selected as human DNMT1, FANCF, RUNX1 gene and EMX1, and the PAM of the target recognition site was TTTV. crRNA expression vectors DNMT1-DFR-D, FANCF-DFR-F2, RUNX1-DFR-R2 capable of simultaneously targeting three genes, and crRNA expression vectors EMX1-EE-E1 and EMX1-EE-E2 capable of simultaneously targeting two genes were designed. The corresponding crRNA sequences are shown in Table 3, and the underlined regions are the targeting regions, which are constructed into crRNA eukaryotic expression vectors.

[0042] Table 3. Name and corresponding sequence of crRNA

[0043] HEK 293T cells were plated at 70-80% confluency, and the number of cells seeded in a 12-well plate was 8 x 10 4 The cells were plated for 6-8 h before transfection. 1 pg of Gs12-7MAX expression plasmid plenti-Gs12-7MAX-puro, Gs12-7EGK expression plasmid plenti-Gs12-7EGK-puro eukaryotic expression vector or known enhanced enAsCas12a-HF1 expression plasmid plenti-enAsCas12a-HF1-puro, 500 ng of crRNA tandem expression vector plenti-U6-DFR-D-DFR-F2-DFR-R2-zsGreen or plenti-U6-EE-E1-EE-E2-zsGreen (as shown in FIG. 3A) that can simultaneously target two or three genes, and 10 pL of Jetprime regent were sequentially added to 200 pL of Jetprime Buffer, mixed well by blowing, and incubated at room temperature for 10 min. The incubated mixture was added to the culture medium of the plated HEK 293T cells for transfection. After 72 h of culture at 37 °C, the culture medium was discarded, the cells were resuspended with 100 pL of PBS, and the genomic DNA of the cells was extracted. The target sites of the positive cells after transfection were PCR-amplified to edit the sequence near the target site. The PCR product was recovered for amplicon sequencing by Illumina Novaseq PE150, and the editing activity was analyzed by CRISPResso2.

[0044] The results are shown in FIG. 3. Gs12-7MAX and Gs12-7EGK nucleases, whether editing two or three sites simultaneously, both have genome editing activity. Without enrichment of gene editing positive cells, the gene editing activity of Gs12-7MAX is as high as about 35%, while that of Gs12-7EGK is only 28% (FIG. 3A). In terms of average gene editing activity, the activity of Gs12-7MAX is significantly higher than that of enAsCas12a-HF1 and Gs12-7EGK, and the activity for simultaneous editing of multiple genes can reach about 20% (FIG. 3B). Therefore, the CRISPR-Gs12-7MAX system has significantly higher gene editing activity.

[0045] Example 4. Detection of Indel distribution characteristics of Gs12-7MAX after site-specific cleavage of target sites

[0046] In this embodiment, high-throughput sequencing and CRISPResso2 software were used to detect the Indel distribution characteristics of Gs12-7MAX, Gs12-7EGK and enAsCas12a-HF1 after target site-directed cleavage. In this embodiment, the target nucleic acid was the gene site of Examples 2 and 3, and the corresponding crRNA is shown in Tables 2 and 3. The PCR products of the above two examples were amplified by Illumina Novaseq PE150 for amplicon sequencing, and the sequencing data were analyzed by CRISPResso2 to further evaluate the distribution and size characteristics of Indels.

[0047] As shown in Figure 4, after the sequencing results of all target sites obtained in Examples 2 and 3 were statistically analyzed, it was further found that the Indel distribution types of Gs12-7MAX, Gs12-7EGK and enAsCas12a were basically consistent (Figure 4A, B, C left side), and most of them were mainly small fragment deletion (about 96%), and a small part of the gene editing sites had small fragment insertion (about 4%). The results showed that after the CRISPR-Gs12-7MAX technology edited the target site, the main base deletion was within 20 bp, and only a small part was within 10 bp (Figure 4A, B, C right side).

[0048] Example 5. Evaluation of the gene editing specificity of Gs12-7MAX and enAsCas12a-HF1

[0049] In this embodiment, crRNA was designed for Gs12-7MAX and enAsCas12a-HF1 site-directed editing in Examples 1 and 2 above, such as 6 crRNAs (named Site1-Site6) of EMX1-2, EMX1-3, EMX1-4, FANCF-2, RUNX1-2 and DNMT1. Using CRISPR-offinder software, 3 off-target sites were predicted for each crRNA as shown in Table 4, which were Site1-offtarget1-1, Site1-offtarget1-2, Site1-offtarget1-3, Site2-offtarget2-1, Site2-offtarget2-2, and 18 off-target sites. The lowercase bases in the sequence in Table 4 are the predicted off-target mismatch sites. After transfecting HEK 293T cells, the predicted off-target sites were PCR amplified and sequenced, and the cleavage efficiency was analyzed by CRISPResso2 software to evaluate the genome cleavage specificity of Gs12-7MAX and enAsCas12a-HF1.

[0050] This example was plated at a confluence of 70-80% in HEK 293T cells, and the number of cells seeded in a 12-well plate was 8 x 10 4 Cells / well. The cells were plated for 6-8 h for transfection, and 1 pg of Gs12-7MAX expression plasmid plenti-Gs12-7MAX-puro or known enhanced enAsCas12a-HF1 expression plasmid plenti-enAsCas12a-HF1-puro, 500 ng of the above different crRNA expression vector plenti-U6-crRNA-zsGreen, and 10 pL of Jetprime regent were sequentially added to 200 pL of Jetprime Buffer, and mixed by blowing, and incubated at room temperature for 10 min. The mixed solution after incubation was added to the culture medium of the HEK 293T cells plated to transfect. After 72 h of culture at 37 °C, the culture medium was discarded, and the cells were resuspended with 100 pL of PBS and the genomic DNA of the cells was extracted. The PCR amplification primers corresponding to the predicted off-target sites were designed as shown in Table 4, and the genomic regions of the candidate predicted off-target sites were amplified by PCR technology. The PCR products were recovered, and amplicon sequencing was performed by Illumina Novaseq PE150, and the cleavage editing activity was analyzed by CRISPResso2 software.

[0051] Table 4. Sequences of predicted candidate off-target sites and corresponding PCR amplification primers

[0052] The results are shown in FIG. 5. High-throughput sequencing found that for the predicted candidate off-target sites, Gs12-7MAX and enAsCas12a-HF1 almost did not detect off-target activity (FIG. 5A). Further comparative analysis found that at multiple predicted off-target sites, no off-target efficiency was detected, and the highest off-target off-target efficiency was less than 1% (FIG. 5B). Overall, Gs12-7MAX has low off-target efficiency, and it has very high specificity as the enhanced and high-fidelity enAsCas12a-HF1 (FIG. 5B). Therefore, Gs12-7MAX can be used as a highly efficient gene editing tool.

[0053] Example 6. High activity and high fidelity characteristics of the CRISPR-Gs12-7MAX system mediated gene editing

[0054] To compare Gs12-7MAX with the known highest activity, high-fidelity AsCas12a Ultra, we transfected plasmids co-expressing Gs12-7MAX or AsCas12a Ultra and their crRNAs into HEK293T cells. Editing efficiency was evaluated at 15 endogenous “TTTV” PAM-containing sites, targeting four genes, DNMT1, FANCF, RUNX1 and VEGFA. The sequences of the corresponding crRNAs are shown in Table 5, with the underlined region being the targeting region, and were constructed into eukaryotic vectors co-expressing Gs12-7MAX / AsCas12a Ultra and crRNAs, respectively.

[0055] Table 5. Names and corresponding sequences of crRNAs

[0056] HEK 293T cells were plated at 70-80% confluency, with 8 x 10 4 Cells were plated for 6-8 h before transfection. 1 pg of plasmids co-expressing Gs12-7MAX or AsCas12a Ultra and their crRNAs, PX330-Gs12-7MAX-crRNA or PX330-AsCas12a Ultra-crRNA, respectively, were added to 200 pL Jetprime Buffer, as shown in FIG. 6A, and 10 pL Jetprime regent was added and mixed well, and incubated at room temperature for 10 min. The mixture was added to the culture medium of the HEK 293T cells for transfection. After 72 h of enrichment with 2.5 pg / mL puromycin antibiotic at 37 °C, the culture medium was discarded, and the cells were resuspended with 100 pL PBS and the genomic DNA was extracted. The target sites of the positive cells were PCR-amplified to sequence the nearby region. The PCR products were recovered for amplicon sequencing by Illumina Novaseq PE150, and the editing activity was analyzed by CRISPResso2.

[0057] As shown in FIG. 6A, both Gs12-7MAX and AsCas12a Ultra have editing efficiency of more than 60% at most sites, and show comparable editing activity at low efficiency (e.g., DNMT1-1, DNMT1-3, FANCF-4) and high efficiency (e.g., DNMT1-4, FANCF-5, RUNX1-6) targets. Notably, the editing activity of Gs12-7MAX at RUNX1-3, VEGFA-1 and VEGFA-2 sites is higher than that of AsCas12a Ultra, which is increased by 11%, 24% and 41% respectively compared with AsCas12a Ultra (FIG. 6A), and the overall comparison of editing activity of 15 sites shows that Gs12-7MAX has comparable editing activity with AsCas12a Ultra with the highest gene editing efficiency (FIG. 6B).

[0058] At the same time, in order to further evaluate the specificity of Gs12-7MAX and AsCas12a Ultra on the whole genome, we performed the GUIDE-seq experiment. Four representative sites, Matched-site-8, DNMT1-site-3, FANCF-target-8 and VEGFA-target-3, were selected. The sequences of the corresponding crRNAs are shown in Table 6, and the underlined region is the targeting region, which was constructed into the eukaryotic vector for co-expression of Gs12-7MAX or AsCas12a Ultra and crRNA.

[0059] Table 6. The name and corresponding sequence of crRNA required for the GUIDE-seq experiment

[0060] HEK 293T cells were plated at a confluence of 70-80%, and the number of cells inoculated in a 6-well plate was 1.6x10 5Cells / well. Cells were plated for 6-8h before transfection, 2μg PX330-Gs12-7MAX-crRNA or PX330-AsCas12a Ultra-crRNA plasmid co-expressing crRNA protein and crRNA and 10pmol double-stranded oligodeoxyribonucleic acid (dsODN) were co-transfected using Lipofectamine 3000 (Invitrogen). After 72h enrichment with 2.5μg / mL puromycin antibiotic at 37℃, the medium was discarded, and the cells were resuspended with 100μL PBS and the genomic DNA of the cells was extracted. The genomic DNA was fragmented to 500bp using NEBNext dsDNA Fragmentase (NEB) kit, followed by end repair, dA tailing and adapter ligation, and the dsODN content fragments were enriched by two rounds of nested anchor PCR. The library was sequenced on Illumina NovaSeq 6000 and analyzed using the GUIDE-seq pipeline (https: / / github.com / aryeelab / guideseq).

[0061] The results are shown in Figure 6C, the target editing activities of the two nucleases at Matched-site-8, DNMT1-site-3, FANCF-target-8 and VEGFA-target-3 were close to 50%, in addition, the double-stranded oligonucleotide (dsODN) insertion rates at these sites were about 0.6% (Figure 6D), which enabled accurate reflection of the actual off-target activity of the two nucleases in the whole genome in GUIDE-seq. At the same time, at FANCF-target-8 and VEGFA-target-3 sites, no off-target production of Gs12-7MAX / AsCas12a Ultra was detected in the whole genome (Figure 6E). And at Matched-site-8 and DNMT1-site-3 sites, Gs12-7MAX produced only 4 and 1 off-target sites in the whole genome, respectively, while AsCas12a Ultra produced 15 and 13 off-target sites in the whole genome, respectively, which indicated that Gs12-7MAX had higher specificity than AsCas12a Ultra. Thus, the CRISPR-Gs12-7MAX system exhibited dual core advantages in mediating gene editing: on the one hand, it had high editing activity and could efficiently complete the editing operation of the target gene; on the other hand, it had high fidelity and could effectively reduce the editing risk of non-target regions, ensuring the precise controllability of the editing process.

[0062] Example 7. The CRISPR-Gs12-7MAX system is suitable for efficient preparation of gene editing animal models

[0063] To evaluate the effect of Gs12-7MAX in vivo gene editing, we tested the editing against mouse tyrosinase (Tyr) gene. The gene is known to encode an enzyme essential for melanin synthesis, and its functional knockout leads to albinism, thus providing a visualized phenotype. The sequences of the corresponding crRNAs designed against Tyr are shown in Table 7, with the underlined region being the targeting region.

[0064] Table 7. Names and corresponding sequences of crRNAs designed to target Tyr gene

[0065] Gs12-7MAX nuclease and two crRNAs targeting Tyr were transcribed using MEGAshortscript T7 Kit (Life Technologies) respectively. The obtained mRNA and crRNAs were purified using MEGAclear Kit and eluted in RNase-free water. Meanwhile, superovulated 8-week-old B6D2F1 female mice were mated with B6D2F1 male mice, and zygotes were collected from oviducts 21 hours after mating. Gs12-7MAX mRNA (50 ng / μL) and Tyr-crRNAs (100 ng / μL) were mixed into droplets in M2 medium and injected into the cytoplasm of zygotes by FemtoJet microinjector (Eppendorf) at a constant flow rate. The injected embryos were cultured overnight in M16 medium supplemented with amino acids at 37°C, 5% CO2, and transferred to the oviducts of pseudopregnant ICR female mice 8 weeks old (Figure 7A).

[0066] The results are shown in Figure 7B. Gs12-7MAX mRNA and two crRNAs specific to Tyr gene were microinjected into D2B6F1 zygotes respectively to test the editing activity of Gs12-7MAX in embryos, and Gs12-7MAX achieved high editing activities of 87.36% and 69.43% respectively against the two target sites of Tyr (Figure 7B). In live-born F0 pups, these rates further increased to 89.89% and 92.33% respectively (Figure 7C). Therefore, Gs12-7MAX effectively achieved gene editing in mouse individuals, and produced a clear albinism phenotype after editing against Tyr gene (Figure 7D). Thus, the CRISPR-Gs12-7MAX system is suitable for efficient preparation of gene-edited mouse models and has broad application prospects in the field of gene therapy and animal model preparation.

[0067] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been described as above with preferred embodiments, it is not intended to limit the present application, and any simple modification, equivalent change and modification of the above embodiments made by those skilled in the art without departing from the technical solution of the present application shall still fall within the scope of the present application.

Claims

1. A nuclease enzyme variant in a CRISPR / Gs12-7 system, characterized in that, The protein comprises: I. a Gs12-7MAX protein having an amino acid sequence as set forth in SEQ ID NO. 1, wherein the amino acid at position 157 is mutated from Glu to Arg, relative to a wild-type Gs12-7 endonuclease; II. a protein having one or more substitutions, deletions, or additions of amino acids, relative to the amino acid sequence as set forth in SEQ ID NO. 1, and substantially retains the biological function derived from the sequence.

2. A fusion protein, characterized in that, The protein of claim 1 and other modified moieties.

3. A polynucleotide, characterized in that, The polynucleotide is a polynucleotide encoding the endonuclease variant of claim 1, or a polynucleotide encoding the fusion protein of claim 2.

4. Vector, characterized in that, The vector comprises the polynucleotide of claim 3.

5. A host cell characterized in that, The host cell comprises the polynucleotide of claim 3 or the vector of claim 4.

6. Use of the endonuclease variant of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the host cell of claim 5, in gene editing.

7. A CRISPR / Gs12-7MAX gene editing system, characterized in that, The endonuclease of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3, or the vector of claim 4, or the host cell of claim 5.

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