Desulfurization vibrio-based I-C type CRISPR gene editing system and application thereof

Abstract

The invention discloses an I-C type CRISPR (clustered regularly interspaced short palindromic repeats) gene editing system based on desulfurization vibrio and application thereof, and belongs to the technical field of gene editing. The gene editing system comprises a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA) piggyBac (PB) vector, an I-C type editor piggyBac vector and a piggyBac (PB) transposase expression vector; the I-C type editor piggyBac vector comprises PB joint sequences at two ends, a Casade component, a Cas3 protein and a Puro resistance gene, and the Casade component is formed by compounding a Cas5c protein, a Cas7c protein, a Cas8c protein and a Cas11c protein, is connected through different 2A peptides and is regulated and controlled by the same CMV promoter. The obtained gene editing system can perform accurate and efficient genome editing and base editing in mammalian cells and animals, defined deletion is realized at a plurality of endogenous sites of human and pig cell lines by utilizing an optimized editor and paired PAM inward crRNAs, and in addition, by combining with an exogenous homologous arm donor vector, the gene editing efficiency is greatly improved, and the gene editing efficiency is improved. And accurate replacement of large gene segments can also be realized.

Description

Technical Field

[0001] The present invention relates to the technical field of gene editing, and in particular to a Desulfovibrio IC-type CRISPR gene editing system and applications thereof. Background Art

[0002] The CRISPR-Cas (Regularly Interspaced Short Palindromic Repeats-CRISPR-associated) system in bacteria and archaea is an adaptive immune system that resists the invasion of exogenous genetic elements. According to their composition and function, they are divided into class I and class II. Currently, effective RNA-guided tools widely used in eukaryotic genome editing, such as type II Cas9, type V Cas12a, and type VI Cas13a, are derived from class I single subunit effectors, which account for only about 10% of all CRISPR-Cas systems. However, the class I multi-subunit effector complex, which occupies about 90%, has not yet been widely developed as a mammalian genome editing tool.

[0003] Type I CRISPR-Cas systems are the most abundant and diverse group of Class I CRISPR systems. According to the composition of the cas operon, the Type I system can be further divided into several subtypes from IA to IG. Generally speaking, the Type I CRISPR system consists of three parts: the guide CRISPR RNA (crRNA), the target recognition module Cascade complex (Cascade), and the DNA cleavage module is usually the Cas3 helicase. Cascade first recognizes and binds to the target sequence adjacent to the 5' adjacent motif (PAM) under the guidance of crRNA, and then forms a stable R-loop, triggering the conformational change of Cascade to recruit the Cas3 helicase, thereby degrading the targeted DNA.

[0004] Compared with the commonly used single effector CRISPR system, the type I CRISPR system has unique characteristics. First, unlike the small fragment deletions and insertions produced by Cas9 and Cas12a, the type I CRISPR-Cas system always cuts the DNA target and causes large fragment deletions; second, in the type I CRISPR system, the target recognition length of crRNA is about 30 bases or longer, which may make it more specific for genome editing than Cas9 and Cas12a; in addition, Cascade, which is composed of multiple proteins, is like a versatile scaffold that can recruit various functional domains for genome manipulation.

[0005] Recently, several type I CRISPR systems have been successfully applied to manipulate the genome of mammalian cells. These include type I Cascade fused to FokI endonuclease from Escherichia coli, which is used to induce target mutations; the IE system from Escherichia coli, which can achieve directional and large-scale DNA degradation in human cells; the ID CRISPR system from Microsporum aeruginosa, which can achieve bidirectional large fragment deletion and small fragment insertion and deletion in plant cells; and the IE, IF, and I-Fv systems from Streptococcus inactivating Trichoderma, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens, which are used to regulate gene expression in plants and humans. All of these systems require the simultaneous overexpression of multiple components to function, and the complexity and relatively large payload size of type I CRISPR systems have hindered their widespread application. Therefore, exploring and simplifying miniature type I CRISPR-Cas systems will provide new tools for genome editing and gene regulation.

[0006] In bacteria, the more streamlined IC-type CRISPR system requires only three unique proteins to form a DNA-targeting Cascade complex. The Cas11c subunit in Cascade is required to safely bind to targeted DNA in mammalian cells, and several IC systems from Neisseria gonorrhoeae, Bacillus halogenus (Bha), and Desulfovibrio (Dvu) have been used for mammalian cell gene editing. However, they can cause uncontrolled large fragments of heterogeneous DNA deletions, which may cause devastating effects. The previously reported IC system from Desulfovibrio showed relatively low DNA cutting activity, but has not yet been fully explored. Summary of the invention

[0007] In order to solve the above technical problems, the purpose of the present invention is to provide a Desulfovibrio IC-type CRISPR gene editing system and its application, so as to solve the problem that the existing IC-type CRISPR system used for mammalian cell gene editing may cause large fragments of heterogeneous DNA deletions and cause destructive effects.

[0008] The technical solution of the present invention to solve the above technical problems is as follows:

[0009] A Desulfovibrio IC-type CRISPR gene editing system includes a crRNA expression vector, an IC-type editor piggyBac vector and a piggyBac transposase expression vector; the IC-type editor piggyBac vector includes PB linker sequences at both ends, a Cascade component, a Cas3 protein and a Puro resistance gene.

[0010] Furthermore, the Cascade component is composed of Cas5c protein, Cas7c protein, Cas8c protein and Cas11c protein, connected by different 2A peptides and regulated by the same CMV promoter.

[0011] Furthermore, the nucleotide sequence of the Cas5c protein is shown in SEQ ID NO.1, the nucleotide sequence of the Cas7c protein is shown in SEQ ID NO.2, the nucleotide sequence of the Cas8c protein is shown in SEQ ID NO.3, and the nucleotide sequence of the Cas11c protein is shown in SEQ ID NO.4.

[0012] Furthermore, the Cas7c protein and the Cas5c protein are connected by F2A, and the nucleotide sequence of F2A is shown in SEQ ID NO.5; Cas5c and the Cas8c are connected by T2A, and the nucleotide sequence of T2A is shown in SEQ ID NO.6; Cas8c and the Cas11c are connected by E2A, and the nucleotide sequence of E2A is shown in SEQ ID NO.7.

[0013] Furthermore, BPNLS is connected to the N-terminus of Cas5c protein, Cas7c protein, Cas8c protein and Cas11c protein; the nucleotide sequence of BPNLS is shown in SE Q ID NO.8.

[0014] Furthermore, the nucleotide sequence of the Cas3 protein is shown in SEQ ID NO.9, the N-terminus of the Cas3 protein is connected to BPNLS, the nucleotide sequence of BPNLS is shown in SEQ ID NO.8, the C-terminus of the Cas3 protein is connected to the nucleoplasmic protein NLS, and the nucleotide sequence of the nucleoplasmic protein NLS is shown in SEQ ID NO.10.

[0015] The above-mentioned Desulfovibrio IC-based CRISPR gene editing system is used for identifying, binding and editing mammalian genes.

[0016] A CRISPR system for identifying, binding and editing mammalian genes, including the above-mentioned Desulfovibrio IC-based CRISPR gene editing system.

[0017] The present invention has the following beneficial effects:

[0018] The present invention optimizes an IC-type CRISPR system from Desulfovibrio vulgaris and demonstrates that it can perform precise and efficient genome editing and base editing in mammalian cells and animals. Using optimized editors and paired PAM-inward crRNAs, the present invention achieves defined deletions at multiple endogenous sites in human and pig cell lines. This is in contrast to the type I system that previously caused uncontrolled and heterozygous editing. The results of the present invention show that the opposite positioning of the Cas complex hinders continuous nuclease activity, thereby achieving restricted cutting between target sites. In addition, using the homology-dependent repair pathway, endogenous DNA can be accurately replaced by paired PAM-in crRNAs. The present invention also demonstrates the efficacy of this platform in primary porcine fibroblasts, and these modified primary fibroblasts of pigs can be successfully used as donor cells to clone genetically engineered pigs containing CD163 SRCR5 domain deletions, which highlights the potential of this tool in agricultural applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Optimization of Dvu IC CRISPR vector and evaluation of DNA cleavage efficiency using reporter system, where A is a schematic diagram comparing the protein sizes of spCas9, AsCas12a, and LbCas12a nucleases widely used in this study and the Dvu I-C CRISPR system in this study, B is a schematic diagram of the optimized humanized Dvu type IC editor piggyBac vector, C is a schematic diagram of the piggyBac vector expressing the pre-crRNA array, R represents the repeat sequence, S1 or S2 represents spacer1 or spacer 2, pT represents the polythymine sequence, D is a schematic diagram of the piggyBac transposase (PBase) expression vector, E is an overview of the HPRT-GFP site in HEK293T cells, where GFP has been integrated, F is the GFP knockout efficiency of Dvu IC editor and spCas9 obtained by flow cytometry analysis, G is a representative image of GFP fluorescence-positive cells after transfection of SSA reporter system vector and CRISPR system with different crRNA or sgRNA, scale bar, 200 μm;

[0020] Figure 2Schematic diagram and experimental results of the IC-type CRISPR system of Desulfovibrio sulphuridis that can achieve efficient and specific genome editing in human cells, where A is an overview of the mini IC-type CRISPRcas gene and CRISPR array in the giant plasmid of Desulfovibrio sulphuridis Hildenborough strain, B is a flowchart of the single-stranded annealing (SSA) reporter experiment, C is the selection of 8 sites to evaluate and compare the single-stranded annealing activity mediated by Dvu IC editor and spCas9 when using a single crRNA / sgRNA to target a 1kb DNA sequence in the SSA reporter plasmid, D is the use of paired crRNAs / sgRNAs to target HPRT, HEK3 and AAVS1 sites, and the SSA reporter system is used to evaluate and compare the site-specific gene deletion editing activity of Dvu IC editor and spCas9, E is an analysis of the DNA cutting efficiency of Dvu IC editor mediated by pre-crRNA or mature crRNA, and D is an analysis of the effect of a single mismatched base pair (red) in the 35nt spacer on the DNA cutting function of Dvu IC editor;

[0021] Figure 3 The experimental results show that the Dvu IC editor can target poly-T-containing DNA targets using pre-crRNA embedded in mRNA, wherein A is a schematic diagram of the SSA reporter experiment (single-strand annealing reporter assay) using crRNA derived from the hU6 promoter or crRNA embedded in the 3'UTR region of the mCherry mRNA transcript to evaluate the gene editing efficiency, B is a representative image of GFP fluorescence-positive cells after transfection of SSA reporter system vectors, Dvu IC editors and different crRNA expression vectors, scale bar, 10 microns, C is the result of analyzing the proportion of GFP-positive cells in each group by flow cytometry after targeting with different crRNAs;

[0022] Figure 4Figure 1. Alignment of deletions induced by the Dvu IC system using paired crRNAs in HEK293T cells and HeLa cells. A is a schematic diagram of the induction of defined deletions by the Dvu-IC editor using the paired PAM-in crRNAs strategy. B and C are long-distance PCR amplifications spanning two cleavage sites. The HPRT, AAVS1, HEK3, and DMD genomic loci in HEK293T cells (B), and the PCSK9 and HEK3 loci in HeLa cells (C) validated the deletions generated by the Dvu IC editor using dual PAM-in crRNA guidance. Each genomic locus was targeted in at least three independent experiments. Untransfected wild-type (WT) cells served as a control. The red dashed box area was purified by gel extraction kit for TA cloning and Sanger sequencing. D is the evaluation of the editing efficiency of the Dvu IC editor using paired PAM-incrRNAs at the HPRT, AAVS1, and HEK3 loci in HEK293T cells by ddPCR. E is an overview of the results of TA cloning based on the Dvu IC editor. Genomic regions excised in engineered alleles at the HPRT, AAVS1, and HEK3 target loci in HEK293T cells and the PCSK9 locus in HeLa cells after editing with PAM-in crRNAs by IC Editor;

[0023] Figure 5The Dvu IC-based CRISPR system with paired PAM-in crRNA showed clear results of DNA deletion in mammalian cells, where A is a schematic diagram of targeted deletion of about 608 bp of DNA sequence in the human HPRT locus by using paired PAM-in crRNAs and the Dvu IC editor, B is a PCR amplification of the region spanning the target site in HEK293T cells, and gel electrophoresis analysis was performed to verify the deletion guided by the Dvu IC editor and the dual PAM-in crRNA, C is a chromatogram of Sanger sequencing of the PCR amplicon after the HPRT locus was deleted by the Dvu IC editor in HEK293T cells, and D is a chromatogram of the HPRT locus in HEK293T cells after the Dvu IC editor was used to delete the HPRT locus. Figure 1: Chromatogram of Sanger sequencing of PCR amplicons after IC editor deletion. E is a clone with modified HPRT gene, the results of genomic DNA extraction, PCR across the target site and gel electrophoresis analysis of the products. F is a schematic diagram of the paired crRNA method, and the PAM-in direction is used to disrupt the B4GALNT2 locus in pigs. G is a schematic diagram of the Dvu type I CRISPR system using paired PAM-in crRNAs to introduce genomic deletions at the GGTA1P locus. H is the result of PCR amplification across the two target sites in PK15 cells to detect deletion events mediated by the Dvu IC editor and paired PAM-in crRNAs targeting the GGTA1P and B4GALNT4 loci, followed by gel electrophoresis analysis. I is an overview of the deleted regions in the GGTA1P and B4GALNT2 loci in PK15 cells after PCR and Sanger sequencing. J is a schematic diagram of the Dvu type I CRISPR system in PK15 cells using paired PAM-in crRNAs. Chromatograms of deleted parts of GGTA1P and B4GALNT2 loci after IC Editor modification after PCR amplification and Sanger sequencing;

[0024] Figure 6The experimental results show that the Dvu IC editor can induce precise deletion of large DNA fragments in mouse liver, wherein A is a schematic diagram of targeted large DNA deletion of the mouse Pcsk9 locus mediated in mouse liver cells by using dual paired PAM-in crRNA and Dvu IC CRISPR system, B is a schematic diagram of transfecting the unitary Dvu IC editor, mouse Pcsk9 PAM-in pre-crRNA array expression vector and CAG-PBase plasmid into mouse liver cells by tail vein high pressure injection, C is the result of PCR amplification and gel electrophoresis across two cleavage sites, which verifies that the Dvu IC editor can use dual PAM-in crRNA to produce deletions in the Pcsk9 genomic locus in mouse liver cells, D is an overview of the fragments deleted by Dvu IC in the Pcsk9 gene in mouse liver cells, which are aligned to the original Pcsk9 locus after PCR and sequencing, and E is an example map of PCR amplification across targets and Sanger sequencing in mouse liver cells after targeted deletion of the Pcsk9 locus by Dvu IC editor;

[0025] Figure 7 With the help of paired PAM-in crRNAs, the Dvu IC CRISPR system can achieve targeted and precise deletion in primary porcine fibroblasts. A is a schematic diagram of the optimized humanized Dvu IC-GFP editor vector, which mainly contains Cascade components, Cas3 protein and eGFP reporter gene cassette. The transfected cells can be sorted by flow cytometry. BD are conceptual diagrams showing the use of dual paired PAM-in crRNA-guided Dvu IC CRISPR system to mediate targeted genomic deletion of CD163, FBN1 and PAX6 loci in primary porcine fibroblasts. EG are overviews of the deleted regions in the allele copies of porcine fibroblast clones after modification of porcine CD163, FBN1 and PAX6 genes, respectively. HJ are maps of PCR amplification and Sanger sequencing of the deleted sites of CD163, FBN1 and PAX6 loci in porcine fibroblast clones after targeted fragment deletion by Dvu IC editor. K is a graph of the Dvu IC Summary of the editing efficiency of CRISPR editors for targeted editing of FBN1, CD163, and PAX6 genes in porcine fetal fibroblasts;

[0026] Figure 8The original porcine fibroblasts transformed by the Dvu IC editor can be used as donors, and the results of the experiment of successfully cloning gene-edited piglets are shown, wherein A is a cloned gene-edited piglet born by CD163 gene knockout mediated by the Dvu IC editor, B is the genotype identification of the cloned pig whose CD163 gene was edited by the Dvu IC editor using a PCR method spanning two target sites, C is the genomic region that was excised in the CD163 locus allele in the transformed piglet, and D is the Sanger sequencing map of the PCR amplified fragment of the CD163 locus deletion in the transformed piglet;

[0027] Fig. 9 The figure shows the results of the endogenous site DNA target knock-in experiment promoted by the Dvu IC CRISPR system in HEK293T cells, wherein A is a schematic diagram of the homology arm donor vector and the homology-dependent insertion strategy of the GFP-CMV-mCherry-IRES-Puro reporter gene mediated by the Dvu IC editor for the HPRT site, HAR or HAL represents the right or left homology arm, respectively, B is the result of flow cytometry analysis, C is the sequence analysis result of the gene replacement cells mediated by the Dvu IC editor, D is a PCR amplification of the region spanning the 5' and 3' homology arms in the HEK293T clone edited at the target site under the mediation of the Dvu IC editor or spCas9 to analyze the replacement of the target fragment, and E is a summary of the percentage of the target fragment replacement mediated by the Dvu IC editor or spCas9 at the HPRT site;

[0028] Fig.10 The experimental results of Dvu Cascade binding to target DNA, where A is a schematic diagram of the Dvu Cascade-VPR vector, B is a schematic diagram of the vector expressing pre-crRNA, C is an overview of the mCherry reporter system (tCherry) for evaluating DNA binding and gene activation efficiency, D is a representative image of mCherry fluorescence-positive cells after transfection of tCherry plasmid, expression vector with target crRNA (On) or expression vector without target crRNA (NT) and DvuCascade-VPR editor, and E is the percentage of mCherry-positive cells tested by FACS analysis;

[0029] Fig.11To achieve efficient A to G conversion in human cells through Dvu-Cascade-mediated ABE, A is a schematic diagram describing different structural designs of Dvu-Cascade-mediated ABE expression vectors, the TadA8e domain is fused to the N-terminus of Cas7c, Cas5c, Cas8c and Cas11c, respectively, and then it is transfected into cells together with crRNA and other Dvu-Cascade subunits, B is a schematic diagram showing the PAM region, the 20-nt region upstream of the PAM, and the 20-nt region downstream of the spacer, C is a comparison of the A to G editing efficiency mediated by Dvu-ABE with different structural designs at two sites in HEK293T cells, D is the frequency of adenine to guanine editing induced by the Dvu-ABE system at each adenine at another 10 endogenous sites in HEK293T cells detected by next-generation sequencing analysis, the PAM sequence is shown in red, and the mean ± standard deviation is from three independent experiments;

[0030] Fig.12 To detect the editing efficiency of A>G after Dvu-ABE editing other endogenous targets using Sanger sequencing and EditR analysis, the results of PCR amplification, Sanger sequencing, and corresponding EditR analysis of 10 endogenous sites using Dvu-ABE in HEK293T cells, with the PAM sequence shown in red and italics;

[0031] Fig.13 Figure 1 is the characteristics of base editing induced by Dvu-CBE. A is the summary of the base editing frequency of each adenine in the spacer region of 12 targets using Dvu-ABE. These data show that the main editing window ranges from positions 10 to 26 in the spacer region. B is the insertion / deletion frequency of the indicated sites in genomic DNA under different conditions. HEK293T cells were treated with Dvu-ABE or nSpCas9-ABE8e, followed by Sanger sequencing and analysis of the sequencing results by Decodr (https: / / decodr.org / analyze). The data represent the average of three independent experiments.

[0032] Fig.14Comparison of the A-to-G base editing efficiency mediated by a monovalent vector and a hybrid vector, A is a schematic diagram of the monovalent Dvu-ABEpiggyBac vector, TadA8e-Cas8c in Dvu-Cascade is driven by a CMV promoter, Cas7c, Cas5c and Cas11c are connected by different 2A peptides and controlled by another CMV promoter, and the Puro resistance gene is driven by the SV40 promoter, B is the A-to-G editing efficiency of the monovalent vector and the hybrid vector at the ASGR1-2, HPRT-2 and HPRT-4 sites in HEK293T cells, the error bars represent three biological replicates, C is the effect of each component in the monovalent Dvu-ABE vector on the A-to-G editing efficiency, and the data represent the average of three independent experiments;

[0033] Fig.15 To achieve efficient C to T conversion in human cells by Dvu-Cascade-mediated CBE, A is a schematic diagram of the Dvu-Cascade-CBEs structure that depicts the fusion of different cytosine deaminases to the N-terminus of Cas8c and the fusion of uracil DNA glycosylase inhibitor (UGI) to the C-terminus of Cas7c. ​​B is the evaluation of the C to T editing efficiency mediated by different Dvu-Cascade-CBE variants at the HPRT-2 locus of HEK293T cells by EditR analysis, with the PAM sequence shown in red. C is the detection of the C to T editing efficiency induced by the Dvu-CBE system at each cytosine on 11 other endogenous target sites, with the PAM sequence marked in red. Cytosines in the GC environment within the spacer sequence (marked with a black triangle) are not easily edited. The mean ± SD is derived from three independent experiments.

[0034] Fig.16 To detect the editing efficiency of C>T after Dvu-CBE editing other endogenous targets using Sanger sequencing and EditR analysis, 12 endogenous sites were edited in HEK293T cells using Dvu-CBE fused to the N-terminus of Cas8c in the Dvu Cascade complex using rAPOBEC1. Results of PCR amplification, Sanger sequencing, and corresponding EditR analysis were performed. The PAM sequence is shown in red and italics. Cytosines in GC sequences in spacer sequences (indicated by black triangles) are not easily edited;

[0035] Fig.17To characterize the base editing induced by Dvu-CBE, after editing 11 targets with Dvu-CBE fused to the N-terminus of Cas8c in the Dvu Cascade complex using rAPOBEC1, the results of the efficiency of each cytosine that underwent base editing in the spacer and its upstream and downstream sequences were summarized. The maximum ratio of cytosine conversion to thymine occurred at three different positions: the third base in the TTC protospacer adjacent motif (PAM), the two bases upstream of the PAM, and the first and second positions of the spacer sequence. It is worth noting that cytosines at positions 11 to 13 and 16 to 21 can also be converted to thymine with relatively high efficiency.

[0036] Fig.18 Off-target analysis of Dvu-BE. A. The DNA cleavage activity of crRNAs of different lengths was evaluated using the SSA reporter system. The SSA reporter vector, Dvu IC editor and crRNAs of different lengths were co-transfected into HEK293T cells, and GFP-positive cells were directly observed by fluorescence microscopy. Scale bar, 100 μm. B. The results of cells analyzed by flow cytometry under the treatment of crRNAs of different lengths as shown in Figure A were statistically analyzed. The data are shown as mean ± SD, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired t-test was used. C. The frequency of A to G base editing of Dvu-ABE on the target site and off-target site at HPRT-1 site and HPRT-2 site, potential off-target The sites are shown on the right. D, the frequency of A to G base editing by spCas9-ABE8e on the target site and off-target site at HPRT-1 and HPRT-2 sites. Potential off-target sites are shown on the right. Mismatched bases in the off-target sequences of Dvu-ABE and spCas9-ABE are marked in purple, while the bases in the gray box, i.e., every 6th position and bases 28 to 35 of the Dvu-Cascade target sequence, are not considered mismatches. The PAM sequence of Dvu-ABE is shown in red italics, and the PAM sequence of spCas9-ABE8e is marked in green italics. The mean ± standard deviation is derived from three independent experiments.

[0037] Fig.19 This is a diagram of the working principle of the present invention. After the PB vector expressing each component of Dvu IC and the vector expressing crRNA are transfected into cells, they will assemble into a complex, thereby exerting different gene editing functions. It can achieve precise deletion of fragments under the action of PAM-incrRNAs; in the presence of homologous arm donor vectors, it can mediate precise replacement of endogenous gene fragments; after modification, a base editing tool that achieves C>T and A>G can be obtained. DETAILED DESCRIPTION

[0038] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0039] Example 1: Genome editing tools based on the Desulfovibrio IC-type CRISPR system exhibit endonuclease activity in human cells

[0040] The Desulfovibrio sulfovibrio (Dvu) IC-type CRISPR system consists of Cascade, Cas3 and crRNA components. The Cascade complex includes Cas5c (227 amino acid residues), Cas7c (290 amino acid residues), Cas8c (612 amino acid residues) and Cas11c (125 amino acid residues).

[0041] The nucleotide sequence of Cas5c protein is shown in SEQ ID NO.1, the nucleotide sequence of Cas7c protein is shown in SEQ ID NO.2, the nucleotide sequence of Cas8c protein is shown in SEQ ID NO.3, and the nucleotide sequence of Cas11c protein is shown in SEQ ID NO.4. Cas7c protein and Cas5c protein are connected by F2A, and the nucleotide sequence of F2A is shown in SEQ ID NO.5; Cas5c and the Cas8c are connected by T2A, and the nucleotide sequence of T2A is shown in SEQ ID NO.6, and Cas8c and the Cas11c are connected by E2A, and the nucleotide sequence of E2A is shown in SEQ ID NO.7. BPNLS is connected to the N-terminus of Cas5c protein, Cas7c protein, Cas8c protein and Cas11c protein; the nucleotide sequence of BPNLS is shown in SEQ ID NO.8. The nucleotide sequence of the Cas3 protein is shown in SEQ ID NO.9, the N-terminus of the Cas3 protein is connected to BPNLS, the nucleotide sequence of BPNLS is shown in SEQ ID NO.8, the C-terminus of the Cas3 protein is connected to the nucleoplasmic protein NLS, and the nucleotide sequence of the nucleoplasmic protein NLS is shown in SEQ ID NO.10.

[0042] Cas5c is responsible for processing pre-crRNA and binding to the crRNA 5'-handle. Cas8c is responsible for recognizing the adjacent sequence adjacent motif (PAM) site on the non-target strand of the double-stranded DNA target. The crRNA is surrounded by a right-handed helical fiber formed by seven Cas7c subunits. The atypical small subunit (Cas11c) stabilizes the formation of the R-loop, which is essential for efficient targeted gene editing in mammalian cells. After the Cascade complex recognizes and binds to the target, the trans-acting nuclease-DNA helicase Cas3 subunit (756 amino acid residues) is recruited and degrades the target DNA ( Figure 2 (Figure A).

[0043] In order to efficiently apply the Dvu IC CRISPR system using plasmids in mammalian cells, the open reading frame (ORF) of each element of the Dvu IC editor was first optimized using human codons, and a dual nuclear localization signal (BPNLS) was added to the N-terminus, which can improve the gene editing activity of Cas9 and base editors. In addition, the present invention removes potential polyA sites (AATAAA or ATTAAA) and Kozak sequences in the synthetic sequence. Then, in order to simplify the process and reduce the number of plasmids used, the present invention designed and constructed a mono-piggyBac transposon plasmid. The vector contains a polycistronic element composed of all Cascade subunit genes driven by a single CMV promoter, and the subunits are connected by various 2A peptides, as well as a Cas3 gene driven by another CMV promoter, and an independent Puro resistance gene expression element box ( Figure 1 crRNA is driven by the U6 promoter and expressed in another piggyBac plasmid ( Figure 1 Under the action of piggyBac transposase, Dvu IC editor and crRNA can be randomly inserted into the genomic TTAA sequence ( Figure 1 D), thereby enabling sustainable editing of targets.

[0044] In order to simply and quickly evaluate the gene knockout activity of the optimized Dvu IC CRISPR system monovector in human cells, the present invention first used flow cytometry to detect the editing efficiency of the GFP fluorescent reporter gene. The crRNA was designed to target the 5'-TTC-adjacent site in the EGFP gene and transfected into the HEK293T GFP reporter cell line together with the Dvu IC editor plasmid. The present invention also transfected the HEK293T reporter cell line with the spCas9 expression vector and the sgRNA expression vector for GFP as a positive control. Flow cytometry analysis showed that the Dvu IC editor exhibited DNA shearing activity ( Figure 1 Figures E and F).

[0045] Then, a single-strand annealing (SSA) recombination assay of GFP was performed to further evaluate the DNA cleavage activity of the Dvu IC-type CRISPR system. The approximately 1kb DNA sequence containing the target site was integrated between the GFP sequences isolated in the pCAG-EGxxFP vector. Another vector expressing BFP driven by the EF1a promoter was used as an internal reference for transfection efficiency. After the reporter plasmid was transfected into HEK293T cells together with the Dvu IC-type CRISPR system or the CRISPR-Cas9 system targeting 8 similar sites of DNA sequences by liposome, the proportion of GFP-positive and BFP-positive cells was evaluated by flow cytometry ( Figure 2 The present invention found that the targeting efficiency of the Dvu IC system and spCas9 varies depending on the specific target ( Figure 2 (C). SSA reporter assays were also used to evaluate and compare the targeted deletion efficiency of Dvu IC editors and spCas9 at HPRT, HEK3, and AAVS1 loci. After transfection of cells and FACS analysis, we observed that the percentage of GFP-positive cells obtained using paired crRNA and Dvu IC editors was comparable to that obtained using two sgRNAs and spCas9 at these loci ( Figure 2 This indicates that the Dvu IC editor can efficiently cleave targeted DNA using either single crRNA or paired crRNA in mammalian cells.

[0046] The DNA cleavage activity of pre-crRNA and mature crRNA was compared using the SSA reporter system. The pre-crRNA contains a 35-nucleotide spacer sequence and two 32-nucleotide repeat sequences. The mature crRNA includes an 11-nucleotide 5' handle structure sequence and a 21-nucleotide 3' hairpin loop structure sequence and a 35-nucleotide spacer sequence. The flow cytometry analysis results showed that the two crRNAs had similar SSA activity ( Figure 2 This is different from the IE-type CRISPR system, which cannot perform gene editing well using mature crRNA of IE-type CRISPR directly. In general, the above experiments show that the Dvu IC-type CRISPR system can achieve efficient DNA cleavage activity in human cells regardless of whether it uses pre-crRNA or mature crRNA.

[0047] To evaluate the targeting specificity of the Dvu IC CRISPR system, the present invention used SSA reporter analysis to evaluate the effect of each single nucleotide mismatch in the 35nt long crRNA on DNA cleavage activity. Flow cytometry analysis showed that any mismatch in the first 5 positions completely abolished DNA cleavage, while single nucleotide mismatches at every 6th position retained considerable activity regardless of the distance from the PAM. It is worth noting that mismatches after position 24 had almost no effect on DNA cleavage ( Figure 2 (Fig. F). The experiment showed that the Dvu IC CRISPR system is very sensitive to mismatches in the seed region close to the PAM, but is more adaptable to mismatches in the distal region of the spacer sequence. This specificity pattern is of great significance for the development of highly specific Dvu IC crRNA for genome editing applications.

[0048] crRNA containing more than 4 consecutive thymidines cannot be efficiently expressed using a pol III promoter (such as the human U6 promoter). If crRNA can be processed from pol II-derived transcripts, it will be beneficial to expand the screening range of crRNA. In addition, by regulating crRNA through a tissue-specific pol II promoter, genes can be edited in specific situations. Furthermore, the Cas5c protein in the DvuI-C system has ribonuclease activity and can process pre-crRNA. In order to explore whether crRNA in pol II-derived transcripts can be processed and effectively edit target genes under the guidance of polyT (pT) crRNA, the present invention introduces two independent Triplex-HPRT pT-crRNA components into the 3'UTR of the mCherry transcript, and uses SSA recombination analysis to evaluate its activity ( Figure 3 As a control, the present invention added two corresponding HPRTpT crRNAs derived from the hU6 promoter. After transfection and flow cytometry analysis, the present invention observed that pT-crRNA1 was unable to mediate target DNA shearing regardless of the promoter used. However, at the second target site, pT crRNA derived from the pol II promoter showed significantly improved editing efficiency ( Figure 3 These results suggest that the Dvu IC editor can efficiently process crRNA embedded in mRNA transcripts and mediate target gene editing.

[0049] Example 2: Dvu IC-mediated DNA deletion region can be restricted by PAM-in crRNA

[0050] The present invention speculates that the Dvu-Cascade-Cas3 ribonucleoprotein complex in opposite directions may hinder the continuous degradation through steric hindrance, thereby limiting the missing region between the two crRNAs ( Figure 4 Figure A in the middle), endogenous human HPRT, AAVS1, HEK3 and DMD genes were selected for testing. A tandem paired PAM-in crRNA expression vector was designed and constructed to delete a 600-700bp DNA fragment. The present invention co-transfected HEK293T cells and HeLa cells with a monovalent Dvu IC piggyBac vector, CAG-PBase and a corresponding paired crRNA vector. After puromycin drug screening enrichment, PCR, agarose gel electrophoresis analysis and Sanger sequencing were performed across two targets, and it was found that targeted deletions of HPRT, AAVS1 and HEK3 were detected in HEK293T cells ( Figure 4 Using digital droplet PCR (ddPCR), the editing efficiencies of HPRT, AAVS1, and HEK3 targets were found to be approximately 54.6%, 46.72%, and 2.89%, respectively ( Fig.15 D in the middle). The DvuI-C editor with paired PAM-in crRNA can also efficiently edit PCSK9 and HEK3 sites in HeLa cells ( Fig.15 The results of long-fragment PCR, TA cloning, and Sanger sequencing showed that the deleted fragments were mainly confined to the region between the two PAM-in crRNAs recognition sequences in both HEK293T cells and HeLa cells ( Figure 4 Figure E). The present invention selected HEK293T cell clones after the HPRT gene was edited by Dvu IC editor and amplified the target gene. The results showed that although all target genes were edited, each clone was heterozygous ( Figure 5 In summary, the Dvu IC CRISPR system can efficiently generate desired specific targeted deletions in human cells using paired PAM-incrRNA.

[0051] To explore the potential of paired crRNA for targeted deletion and its applicability in different in vitro and in vivo cell types, the feasibility of the Dvu IC system using paired PAM-in crRNA to target four different sites in the porcine B4GALNT2 and GGTA1P genes was evaluated ( Figure 5 After electro-transfection and puromycin drug screening, PCR amplification and agarose gel electrophoresis analysis were performed across the target sites ( Figure 5 Figure 3) and Sanger sequencing analysis ( Figure 5In Figures I and J), the present invention detected a DNA fragment of 1772 bp missing from the B4GALNT2 gene and a DNA fragment of 2017 bp missing from the GGTA1P gene in the pig PK15 cell line. In addition, by high-pressure intravenous injection of a single Dvu IC piggyBac editor, CAG-PBase and paired PAM-inPcsk9 crRNAs, the present invention was also able to remove an 11.38 kb fragment of the Pcsk9 gene in the mouse liver ( Figure 6 These results demonstrate the ability of the Dvu IC system to achieve targeted deletion in different cell types.

[0052] Example 3: Efficient defined deletion in primary cells using paired PAM-in crRNA

[0053] Gene editing in primary cells is essential for various applications, including gene therapy, cell therapy, and animal disease model construction. To this end, the present invention detects the editing efficiency of the Dvu IC system in primary porcine fibroblasts mediated by crRNA in the form of paired PAM-in. In order to minimize the potential damage of antibiotics to primary cells and optimize the workflow of the DvuI type C system, the present invention replaces the puromycin resistance gene (called Dvu IC-GFP editor) with the EGFP gene, so that transfected cells can be separated by flow cytometry (FACS) without puromycin selection or random integration by piggyBac transposon ( Figure 7 The present invention targets porcine CD163, FBN1 and PAX6 genes to evaluate the efficiency of deleting 807 bp, 1314 bp and 4353 bp fragments, respectively, in porcine fibroblasts ( Figure 7 B-D in the middle). Mutations in exon 64 of the FBN1 gene cause its premature termination, resulting in C-terminal truncation and deficiency of fibrin-1 protein, which can cause Marfanoid-progeroid-lipodystrophy syndrome. Deletion of the CD163 SRCR5 domain can inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication, and a 4.25 kb heterozygous deletion containing the 5' region of the PAX6 gene may cause congenital iris loss.

[0054] After transfecting Bama porcine fetal fibroblasts (PFFs) with plasmids expressing Dvu I C-GFP editor and PAM-in crRNA array components and sorting out positive cells by flow cytometry, the present invention selected and amplified 30 FBN1 gene clones, 25 CD163 gene clones and 39 PAX6 gene clones. Among these clones, 9 FBN1 clones, 3 CD163 clones and 2 PAX6 clones were confirmed to be successfully edited by the Dvu I C system ( Figure 7 The deletion positions of all fragments were limited to the recognition sequences of the pair of crRNAs. Among them, 8 FBN1 clones were heterozygous, and only 1 carried a homology deletion in the intron ( Figure 7 All four CD163 clones and two PAX6 clones were heterozygous ( Figure 7 The present invention uses CD163 gene-edited cells for somatic cell nuclear transplantation (SCNT) to study their developmental capacity. The present invention obtained 4 piglets ( Figure 8 PCR and Sanger sequencing confirmed that the three CD163-edited piglets carried the same expected deletion ( Figure 8 In summary, the above results demonstrate that the Dvu IC CRISPR system was successfully used for gene editing in primary porcine fibroblasts and to obtain genetically modified cloned piglets. These findings pave the way for the widespread application of other primary cells.

[0055] Example 4: Accurate and efficient gene replacement using paired PAM-in crRNA

[0056] The present invention uses PAM-in crRNA, which has been previously verified to have targeting efficiency in HEK293T cells, to perform experiments and replace the approximately 600 bp DNA fragment in the human HPRT gene with the EGFP-CMV-Puro-IRES-mCherry gene fragment ( Fig. 9 (A). After transfection and puromycin selection, the percentage of GFP-positive cells in different groups was assessed by flow cytometry.

[0057] Compared with the control group without CRISPR system, both Dvu IC editor and spCas9 significantly improved the efficiency of targeted integration ( Fig. 9 Cross-homologous arm PCR and Sanger sequencing further confirmed that Dvu IC can mediate precise targeted replacement ( Fig. 9In order to more accurately evaluate the efficiency of large fragment replacement, the present invention picked the puromycin-resistant clones from Dvu IC editor and spCas9 and performed PCR genotyping analysis ( Fig.18 The results showed that the ratios of correctly replaced clones to total clones in the Dvu IC and spCas9 systems were: Dvu IC editor 12 / 65 (18.46%), spCas9 15 / 43 (34.88%) ( Fig. 9 These results demonstrate that the Dvu IC system can achieve precise gene replacement in human cells by utilizing paired PAM-in crRNA and homology-dependent repair pathways, which is important for inserting reporter genes or repairing disease-causing mutations.

[0058] Example 5: Transforming the Dvu IC CRISPR System into a Gene Activator and Adenine Base Editor

[0059] The Dvu-Cascade effector complex without the Cas3c subunit is responsible for DNA targeting and binding, similar to the inactivated spCas9. In addition, the size of Dvu-Cascade is comparable to that of spCas9, AsCas12a, and LbCas12a ( Figure 1 Figure A-Figure B and Figure 2 To verify the DNA binding ability of Dvu-Cascade, the present invention designed a Dvu-Cascade-VPR (VP64-p65-Rta AD) activation platform by fusing the VPR transcription activator to the C-terminus of Cas7c in the Dvu-Cascade structure ( Fig.10 Then, the present invention uses a reporter gene system to test the gene activation effect. The reporter vector contains a DvucrRNA binding site, followed by a basic minimal CMV promoter and an mCherry expression cassette (named tCherry reporter vector) ( Fig.10 The present invention uses the Dvu-Cascade-VPR system vector and the corresponding crRNA expression vector ( Fig.10 After co-transfection of the tCherry reporter plasmid into HEK293T cells, high expression of mCherry could be observed by fluorescence microscopy. In contrast, no obvious mCherry signal was observed in the control transfection group cells using non-targeted (NT) crRNA ( Fig.10 Flow cytometry analysis further confirmed that the Dvu-Cascade-VPR complex effectively binds to target DNA and stimulates gene expression ( Fig.10 (Fig. E).

[0060] Next, the present invention transformed the Dvu IC CRISPR system into an adenine base editor suitable for human cells. To find the structure with the highest base editing efficiency, the present invention fused the evolved TadA8e deaminase to the N-terminus of different subunits (Cas5c, Cas7c, Cas8c or Cas11c) in the Dvu-Cascade structure and evaluated their base editing efficiency ( Fig.11 Sanger sequencing and EditR analysis of A to G transitions at HPRT-1 and HPRT-2 loci in HEK293T cells showed that ligation of TadA8e to the N-terminus of Cas8c resulted in the best editing ( Fig.11 The present invention named this optimal structure Dvu-ABE. Then, the present invention used Sanger sequencing, EditR analysis and next-generation sequencing (NGS) to test the editing efficiency of Dvu-ABE at other endogenous genomic sites containing TTC PAM. At all tested sites, Dvu-ABE successfully induced A to G conversion, with the highest single A to G frequency ranging from 10% to 61.63% ( Fig.11 D Figure and Fig.12 The main editing window is between +10 and +26 of the interval sequence ( Fig.13 Interestingly, at the VEGFA-1 target site, the present invention also detected an A to G change 2 bases upstream of the PAM and an efficient T to C change at the third base upstream ( Fig.11 This is probably due to the deamination of an A on the opposite side chain by TadA8e. In addition, the Dvu-ABE system without Cas3c nuclease does not generate unwanted indels ( Fig.13 Together, these results demonstrate that Dvu-ABE can safely and efficiently mediate A to G base editing at endogenous genomic loci in human cells.

[0061] In order to reduce the number of plasmids required for transfection, the present invention assembles the various elements of Dvu-ABE into a single vector. In this vector, TadA8e-Cas8c expression is driven by a CMV promoter, while Cas5c, Cas11c and Cas7c are linked by a 2A peptide and driven by a second CMV promoter. In addition, a puromycin resistance cassette ( Fig.14 The present invention uses Sanger sequencing and EditR analysis to compare the A-to-G editing efficiency of a monovalent vector and a separate multiple Dvu-ABE element vector at the ASGR1-2, HPRT-2, and HPRT-4 sites. The editing efficiency of the monovalent vector is lower than that of the mixed plasmid combination at all three tested sites ( Fig.14However, further analysis of MALAT1 A-to-G editing showed that overexpression of Cas7c, TadA8e-Cas8c, or the entire monolithic vector enhanced the A-to-G mutation ( Fig.14 (C). These results highlight the key role of Cas7c and TadA8e-Cas8c in adenine base editing. The optimized monobasic Dvu-ABE vector can simplify and streamline adenine base editing experiments.

[0062] Example 6: Transforming the Dvu IC CRISPR System into a Cytosine Base Editor

[0063] Dvu-ABE successfully mediated A to G base editing, which inspired the present invention to further transform the IC system into a cytosine base editor. In order to optimize the cytosine base editing mediated by the Dvu IC system, the present invention replaced the TadA8e domain in Dvu-ABE with various cytosine deaminases, including the newly discovered TadA8e-N46L (Chen et al., 2023), TadA-CDd (Neugebauer et al., 2023) and TadA-Dual (Neugebauer et al., 2023), as well as the commonly used rat apolipoprotein B mRNA editing enzyme catalytic subunit 1 (rAPOBEC1) (rA1) (Koblan et al., 2018), and Anc689 APOBEC (Koblan et al., 2018). The present invention also fused two UGIs to the C-terminus of Cas7c. ​​Since there are 7 Cas7 subunits in Cascade, about 14 UGIs can be produced in the Dvu-Cascade complex ( Fig.15 Then, the present invention first compared the C to T editing efficiency of these variants at the endogenous HPRT-2 site of HEK293T cells. From the results of Sanger sequencing and EditR analysis, rA1 and Anc689 APOBEC performed better than other cytosine deaminases ( Fig.15 The present invention further tested the editing efficiency of the Dvu-rA1 CBE system at 12 additional endogenous sites containing TTC PAM sequences by Sanger sequencing, EditR analysis, and NGS experiments. At all 12 sites, Dvu-rA1 CBE successfully induced C to T conversion, with the highest single C to T frequency ranging from 11.5% to 57.08% ( Fig.15 Figure C and Fig.16 ). Consistent with previous studies, rA1 in the Dvu-rA1CBE system hardly induces C to T conversion at the cytosine following guanine ( Fig.15 Figure C and Fig.16After removing these sites that do not meet the conditions, the present invention summarizes the efficiency of C-to-T conversion at these sites and finds that the editing window of Dvu-CBE is mainly distributed in three regions. The highest C-to-T conversion is located at the third base of the TTC PAM sequence, 2 bases upstream of the PAM, and the 1st and 2nd positions of the spacer sequence. Cytosine from the 11th to 13th bases of the spacer sequence and from the 16th to 21st bases can also be converted to thymine with moderate efficiency ( Fig.17 Interestingly, at certain target sites, the present invention also detected a G-to-C change upstream of the PAM ( Fig.15 Middle C and Fig.16 ), which may be caused by the deamination of one C on the opposite side chain by rA1. Taken together, these results indicate that Dvu-CBE can efficiently mediate C-to-T editing of endogenous genomic sites with TTC PAM in human cells.

[0064] Example 7: Off-target analysis of the Dvu-BE system

[0065] The long spacer sequences of the Dvu IC system are naturally highly specific. Next, the present invention attempts to analyze the off-target effects of the Dvu I-CCRISPR system. Since the long spacer sequences make it difficult to find off-target sites with low mismatches, the first goal of the present invention is to determine the minimum crRNA length that can mediate gene editing with Dvu IC. To this end, the present invention co-transfected crRNAs of various lengths with Dvu IC and SSA reporter genes into HEK293T cells. The results of the present invention show that crRNAs of 28-nt to 35-nt can be successfully edited, while crRNAs of 24-nt and 26-nt cannot be effectively edited ( Fig.18 Figures A and B in the figure). This shows that mismatches at bases 29 to 35 are more tolerable than mismatches at other bases. In addition, the present invention found that mismatches at positions 1-5 completely eliminated DNA cleavage activity, while mismatches at every 6th base of Dvu crRNA had less effect, allowing gene editing ( Figure 2 Based on these findings, the present invention uses 28-nt crRNA to investigate potential off-target sites, ensuring that positions 1-5 are fully matched and allowing mismatches at positions 6, 12, 18, 24, and 29-35. The present invention identifies potential off-target sites with less than 5 mismatches with Dvu crRNA ( Fig.18 Figures C and D).

[0066] Since the Dvu IC editor will produce uncontrollable products after cutting the genome, it is more convenient to use Dvu-BE to analyze the off-target effects of the Dvu IC system. To this end, the present invention selected two overlapping target sites of Dvu-ABE and nSpCas9-ABE8e in the HPRT gene for off-target analysis. The potential off-target sites of nSpCas9-ABEe were selected with less than 4 mismatches. Then, the present invention evaluated the A to G conversion efficiency of the two target sites and their potential off-target sites. As expected, no obvious off-target A to G base editing was detected in all potential off-target sites of Dvu-ABE ( Fig.18 However, nSpCas9 showed significant off-target effects at HPRT-1OT1, HPRT-2OT3, and HPRT-2OT4 ( Fig.18 In summary, the present invention believes that the Dvu IC system has a high degree of natural specificity in human cells.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A CRISPR gene editing system based on Desulfovibrio IC type, characterized by: It includes a crRNA expression vector, an IC-type editor piggyBac vector and a PB transposase expression vector; the IC-type editor piggyBac vector includes PB linker sequences at both ends, a Cascade component, a Cas3 protein and a Puro resistance gene.

2. The Desulfovibrio IC-type CRISPR gene editing system according to claim 1, characterized in that: The Cascade component is composed of Cas5c protein, Cas7c protein, Cas8c protein and Cas11c protein, which are connected by different 2A peptides and regulated by the same CMV promoter.

3. The Desulfovibrio IC-type CRISPR gene editing system according to claim 1, characterized in that: The nucleotide sequence of the Cas5c protein is shown in SEQ ID NO.1, the nucleotide sequence of the Cas7c protein is shown in SEQ ID NO.2, the nucleotide sequence of the Cas8c protein is shown in SEQ ID NO.3, and the nucleotide sequence of the Cas11c protein is shown in SEQ ID NO.

4.

4. The Desulfovibrio IC-type CRISPR gene editing system according to claim 1, characterized in that: The Cas7c protein and the Cas5c protein are connected via F2A, and the nucleotide sequence of F2A is shown in SEQ ID NO.5; the Cas5c and the Cas8c are connected via T2A, and the nucleotide sequence of T2A is shown in SEQ ID NO.6; the Cas8c and the Cas11c are connected via E2A, and the nucleotide sequence of E2A is shown in SEQ ID NO.

7.

5. The Desulfovibrio IC-based CRISPR gene editing system according to claim 1, characterized in that: The N-termini of the Cas5c protein, Cas7c protein, Cas8c protein and Cas11c protein are all connected to BPNLS; the nucleotide sequence of BPNLS is shown in SEQ ID NO.

8.

6. The Desulfovibrio IC-type CRISPR system according to claim 1, characterized in that: The nucleotide sequence of the Cas3 protein is shown in SEQ ID NO.9, the N-terminus of the Cas3 protein is connected to BPNLS, the nucleotide sequence of BPNLS is shown in SEQ ID NO.8, the C-terminus of the Cas3 protein is connected to the nucleoplasmic protein NLS, and the nucleotide sequence of the nucleoplasmic protein NLS is shown in SEQ ID NO.

10.

7. Use of the Desulfovibrio IC-type CRISPR gene editing system according to any one of claims 1 to 6 in identifying, binding and editing mammalian genes.

8. A CRISPR system for recognizing, binding and editing mammalian genes, characterized in that: Including the Desulfovibrio IC type CRISPR gene editing system as described in any one of claims 1-6.

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