WO2023165613A1 - Use of 5'→3' exonuclease in gene editing system, and gene editing system and gene editing method - Google Patents

Abstract

A use of a 5'→3' exonuclease in a gene editing system, and the gene editing system and a gene editing method. The 5'→3' exonuclease and a site-specific nuclease in the gene editing system are in a non-fusion state; and the gene editing system comprises the site-specific nuclease, the 5'→3' exonuclease, and a donor DNA, wherein the 5'→3' exonuclease and the site-specific nuclease are in a non-fusion state.

Description

Use of 5'→3' exonuclease in gene editing system and gene editing system and editing method thereof technical field

The invention relates to the field of biotechnology, in particular to a use of a 5'→3' exonuclease in a gene editing system, a gene editing system and an editing method thereof.

Background technique

Gene editing technology changes the expression and function of proteins by modifying the gene sequence, including the insertion, deletion and substitution of nucleic acid bases, thereby affecting various traits of organisms. In the field of biotechnology, the use of gene editing technology to change specific nucleic acid sequences, optimize the existing functions of proteins or generate new functions, so as to enhance the traits of organisms, has become the basis of biological industries such as biological breeding. In the field of biomedicine, the use of gene editing technology to repair specific site mutations to normal sequences may be the only effective way to completely cure genetic diseases. Therefore, gene editing technology (precision gene editing technology), which can efficiently and accurately generate insertion, deletion and replacement of specific nucleic acid bases, is a key technology in important fields such as bioindustry and biomedicine. Among them, improving homologous recombination repair ( HDR) efficiency and reduction of non-homologous end-joining repair (NHEJ) are key technical requirements for precise gene editing.

Therefore, there is an urgent need for a precise gene editing system that improves HDR efficiency.

Contents of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent.

To this end, the present invention provides a use of a 5'→3' exonuclease in a gene editing system, a gene editing system and an editing method thereof, by introducing the 5'→3' exonuclease into the gene editing system , can improve homologous recombination repair (HDR) efficiency, reduce non-homologous end-joining repair (NHEJ), so as to realize precise gene editing. It should be noted that the present invention is completed based on the following work of the inventors:

The precision gene editing technology consists of two relatively independent parts: the first part is to identify and cut the target gene site through nuclease, and create nucleic acid damage near the site to be edited; the second part is to use the endogenous nucleic acid damage of the cell The repair process introduces specific nucleic acid changes. The technical differences of precision gene editing technology are concentrated in the second part, in which the main technical routes include oligonucleotide editing template-mediated homologous recombination (ssODN HDR), single base editing (Base Editing, BE), guided editing ( Prime Editing, PE) and double-stranded long-chain nucleic acid editing template-mediated homologous recombination (dsDNA HDR), etc. The above technical route has relatively clear limitations: (1) The editing range is small, and ssODN HDR, BE, and PE are mainly edited near nucleic acid damage (within 10 bases), so most genomic sites cannot be effectively edited by this method Editing; Among them, BE can only introduce very limited types of base changes in addition to the small editing range, so it is limited to gene editing applications in special cases; (2) The efficiency is low, although the dsDNA HDR technology route has the ability to realize genome-wide sites However, this method is extremely inefficient and accompanied by uncontrollable random gene mutation (non-homologous recombination, NHEJ), which limits its practical use.

Based on this, the inventors found through a large number of experiments that the exonucleic acid excision efficiency in the 5'→3' direction of the nucleic acid damage site is the main factor limiting the editing efficiency in the dsDNA HDR technical route; and the non-mammalian endogenous 5'→3 Exonucleases in the 'direction, especially those derived from the 5'→3' direction of bacteriophage, have a wide range of highly active in vivo activities. The inventor also found through experiments that during the gene editing process, the efficiency of dsDNA HDR can be significantly improved by exogenously expressing and recruiting the exonuclease in the 5'→3' direction of the phage to the target gene editing site, and at the same time greatly Inhibition of NHEJ.

In one aspect of the present invention, the present invention proposes the use of a 5'→3' exonuclease in a gene editing system. According to an embodiment of the present invention, the 5'→3' exonuclease is in a non-fused state with the site-specific nuclease in the gene editing system.

In the second aspect of the present invention, the present invention provides a gene editing system. According to an embodiment of the present invention, the gene editing system includes: a site-specific nuclease, a 5'→3' exonuclease, and a donor DNA; wherein, the 5'→3' exonuclease and the The site-specific nucleases are non-fused.

In the third aspect of the present invention, the present invention provides a method for gene editing of cells. According to an embodiment of the present invention, the method includes: introducing the above-mentioned gene editing system into cells.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and understandable from the description of the embodiments in conjunction with the following drawings, wherein:

Fig. 1 is a schematic diagram of the working principle of the TLR reporting system in the embodiment of the present invention;

Figure 2 is a histogram of gene editing efficiency in Example 1 of the present invention;

3 is a histogram of gene editing efficiency in Example 2 of the present invention;

Figure 4 is a histogram of gene editing efficiency in Example 3 of the present invention;

Figure 5 is a histogram of gene editing efficiency in Example 4 of the present invention;

Figure 6 is a protein labeling map of the cell NPM1 site in Example 5 of the present invention;

Figure 7 is a protein labeling map of the cell FUS site in Example 5 of the present invention;

8 is a schematic diagram of the editing system of PE-T7 exonuclease in Example 6 of the present invention;

Fig. 9 is a histogram of editing efficiency of the PE system in Embodiment 6 of the present invention;

Figure 10 is a histogram of HDR pathway repair efficiency with/without MS2 recruitment system in Example 7 of the present invention;

11 is a heat map of different lengths of single-stranded DNA produced by T7 exonuclease at DSB in the cell in Example 8 of the present invention;

Figure 12 is a histogram of gene editing efficiency at the HEK3 locus of the Hela cell line in Example 9 of the present invention;

Figure 13 is a histogram of gene editing efficiency at the DNMT1 locus of the Hela cell line in Example 9 of the present invention;

Figure 14 is a histogram of gene editing efficiency at the HEK3 locus in the HCT116 cell line in Example 9 of the present invention;

Figure 15 is a histogram of gene editing efficiency at the DNMT1 locus of the HCT116 cell line in Example 9 of the present invention;

Figure 16 is a histogram of gene editing efficiency at the HEK3 locus in the U2OS cell line in Example 9 of the present invention;

Figure 17 is a histogram of gene editing efficiency at the DNMT1 locus of the U2OS cell line in Example 9 of the present invention;

Figure 18 is a histogram of gene editing efficiency at the mESC cell line EMX1 site in Example 9 of the present invention;

Figure 19 is a histogram of editing efficiency at the EMX1 site in rat primary neurons in Example 10 of the present invention;

Figure 20 is a histogram of gene editing efficiency at TLR sites in Example 11 of the present invention;

Figure 21 is a histogram of gene editing efficiency at the HEK3 locus in Example 11 of the present invention;

Figure 22 is a histogram of gene editing efficiency at the DNMT1 site in Example 11 of the present invention;

Figure 23 is a histogram of gene editing efficiency at the RNF2 locus in Example 11 of the present invention;

Figure 24 is a histogram of gene editing efficiency at the VEGFA site in Example 11 of the present invention;

Figure 25 is a histogram of gene editing efficiency at TLR sites in Example 12 of the present invention;

Figure 26 is a histogram of gene editing efficiency at the HEK3 locus in Example 12 of the present invention;

Figure 27 is a histogram of gene editing efficiency at the DNMT1 site in Example 12 of the present invention;

Figure 28 is a histogram of gene editing efficiency at the RNF2 site in Example 12 of the present invention;

Figure 29 is a histogram of gene editing efficiency at the VEGFA site in Example 12 of the present invention;

Figure 30 is a histogram of gene editing efficiency at TLR sites in Example 13 of the present invention;

Figure 31 is a histogram of gene editing efficiency at the HEK3 locus in Example 13 of the present invention;

Figure 32 is a histogram of gene editing efficiency at the DNMT1 site in Example 13 of the present invention;

Figure 33 is a histogram of gene editing efficiency at the RNF2 locus in Example 13 of the present invention;

Figure 34 is a histogram of gene editing efficiency at the VEGFA site in Example 13 of the present invention;

Figure 35 is a histogram of gene editing efficiency in the introduction of insertion mutations in Example 17 of the present invention;

Fig. 36 is a histogram of gene editing efficiency in the introduction of replacement mutations in Example 17 of the present invention;

Figure 37 is a graph showing the result of NPM1 double-color marking in Example 14 of the present invention;

Figure 38 is a diagram of the results of NPM1 double-color labeling immunofluorescence verification in Example 14 of the present invention;

Fig. 39 is a histogram of high-throughput screening results in Example 15 of the present invention;

Figure 40 is a representative result diagram of the high-throughput screening results in Example 15 of the present invention;

Figure 41 is a diagram of the results of immunofluorescence verification of the high-throughput screening results in Example 15 of the present invention;

Figure 42 is a diagram of the verification results of polyQ editing in Example 16 of the present invention;

Fig. 43 is a histogram of the effect of polyQ length on ATXN2 stress response in Example 16 of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below. The embodiments described below are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

It should be noted that the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of indicated technical features. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

The term "5'→3' exonuclease" as used herein refers to an exonuclease that degrades DNA from the 5' end, ie in the 5' to 3' direction. The 5'→3' exonuclease of interest can remove nucleotides from the 5' end of a dsDNA strand at blunt ends and, in certain embodiments, at 3' and/or 5' overhangs.

The term "non-fusion state" used herein refers to the independent existence of the 5'→3' exonuclease and the site-specific nuclease in the gene editing system, without any connection relationship between the two, Linkages include linkages to regulatory elements (eg, but not limited to, promoter sequences, transcription termination sequences, etc.), nucleic acid sequences (eg, coding sequences or open reading frames) and/or linkages via peptide chains/covalent bonds. In other words, the final functional state of the 5'→3' exonuclease and site-specific nuclease used herein does not constitute a fusion protein.

As used herein, the terms "gRNA" and "guide RNA" are used interchangeably and refer to a gene that is capable of forming a complex with a CRISPR nuclease and targeting the complex to a target sequence due to a certain complementarity to the target sequence. sequence of RNA molecules. For example, in a Cas9-based gene editing system, the gRNA is usually composed of partially complementary crRNA and tracrRNA molecules that form a complex, where the crRNA contains sufficient identity to the target sequence and guides the CRISPR complex (Cas9+crRNA+tracrRNA) to the target sequence. target sequence A sequence to which a sequence specifically binds. However, it is known in the art that single-guide RNAs (sgRNAs) can be designed that contain features of both crRNAs and tracrRNAs. In the Cas12a-based gene editing system, the gRNA is usually only composed of mature crRNA molecules, where the crRNA contains a sequence that has sufficient identity with the target sequence and guides the complex (Cas12a+crRNA) to specifically bind to the target sequence. It is within the purview of those skilled in the art to design appropriate gRNA sequences based on the CRISPR nuclease used and the target sequence to be edited. It is known in the art that pegRNA can be designed. PegRNA is a derivative form of sgRNA. A short RNA sequence extends from the 3' end of sgRNA. The edited target sequence (if used for deletion, there is no intermediate sequence) is generally suitable for PE editing system, and can be used as a replication template for reverse transcriptase while performing the basic functions of sgRNA.

As used herein, the term "recruitment system" refers to a system that enriches more than one protein into another protein or region of interest. The recruitment system involves the enrichment of multiple one protein to another target protein or target region through protein-protein interaction, protein-small molecule interaction or protein-nucleic acid interaction through non-covalent intermolecular force system; the recruitment system may also include a system for covalently linking multiple proteins to another target protein or target region through chemical modification. Commonly used protein-protein interactions such as the suntag system; protein-nucleic acid interactions such as MS2-MCP, PP7-PCP systems, etc.

The term "genome" as used herein encompasses not only chromosomal DNA present in the nucleus but also organelle DNA present in subcellular components of cells such as mitochondria.

The term "animal cell" as used herein includes cells of any animal body suitable for genome editing. Examples of animal bodies include, but are not limited to, mammals such as humans, mice, rats, monkeys, dogs, pigs, sheep, cows, and cats; poultry such as chickens, ducks, and geese.

The present invention proposes a use of a 5'→3' exonuclease in a gene editing system, a gene editing system and an editing method thereof, which will be described in detail below.

Use of 5'→3' exonuclease in gene editing system

In one aspect of the present invention, the present invention proposes the use of a 5'→3' exonuclease in a gene editing system. According to an embodiment of the present invention, the 5'→3' exonuclease is in a non-fused state with the site-specific nuclease in the gene editing system.

HDR pathway repair is the most accurate repair pathway in the gene editing system. Compared with other repair pathways, the HDR pathway is more suitable for gene therapy repair pathways, but no efficient gene editing tools have been developed yet. The reason is that the HDR repair pathway is only active in the S/G2 phase of the cell cycle, and is inhibited by various regulatory mechanisms in other phases of the cell, but even in the S/G2 phase, NHEJ will compete with the HDR pathway. And it was found through experiments that the gene editing based on the HDR pathway using Cas9 has only about 1% efficiency in cell lines, and HDR repair hardly occurs in non-dividing cells without a cell cycle. Therefore, for such a low efficiency, it cannot be used as an experimental tool, let alone a treatment.

Based on this, the inventors, through in-depth research on gene editing technology, found that the exonucleic acid excision efficiency in the 5' direction of the nucleic acid damage site is the main factor that limits the editing efficiency in the technical route of the gene editing system; and, non-mammalian endogenous Exonuclease in the 5' direction, has a wide range of highly active in vivo activities.

Specifically, the inventors found through experiments that in the process of gene editing, through exogenous expression and recruitment of 5'→3' exonuclease to the target gene editing site, the 5'→3' exonuclease can double DNA DNA double strand break (DNA Double Strand Break, DSB) is cleaved at the end to supplement/replace the cleavage of endogenous nucleases in cells, which can increase the production of long fragments of single-stranded DNA in the middle state of the HDR pathway, thereby improving the repair efficiency of the HDR pathway , while greatly inhibiting NHEJ.

At the same time, the inventors found through RNA sequencing analysis of dividing cells (such as HEK293 cells) and non-dividing cells (such as nerve cells), that for both cell types, the related factors of the NHEJ repair pathway are highly expressed. However, the HDR pathway occurs almost only in dividing cells, and non-dividing cells lack CtIP and other proteins required to initiate the end-cleavage step of the HDR pathway, resulting in the absence of the HDR pathway in non-dividing cells. Moreover, the inventors found that in non-dividing cells, there is a certain expression level of proteins in steps such as insertion and replication of the downstream chain of the HDR pathway. Cleavage of the ends can be accomplished, and the HDR pathway can be induced in non-dividing cells.

Therefore, the inventors improved the competitiveness of HDR pathway repair relative to NHEJ repair by adding 5'→3' exonuclease to the gene editing system. HDR pathway repair also occurs in other periods, thereby improving the efficiency of HDR pathway repair, while greatly inhibiting NHEJ; and, the present invention also found that through the addition of 5'→3' exonuclease, it can be achieved in non-dividing cells HDR pathway.

It should be noted that the use of 5'→3' exonuclease in gene editing system is the use of 5'→3' exonuclease in gene editing. In other words, it refers to a A method for gene editing, the method comprising providing a 5'→3' exonuclease, and using the 5'→3' exonuclease for gene editing.

According to an embodiment of the present invention, the cells used in the gene editing system are animal cells, preferably mammalian cells. The inventors have found through a large number of experiments that the repair efficiency of the HDR pathway can be improved by adding 5'→3' exonuclease, which is suitable for the gene editing system of animal cells, especially for mammalian cells.

According to an embodiment of the present invention, the 5'→3' exonuclease is T7 exonuclease.

Through a large number of experiments, the inventors compared various 5'→3' exonucleases (referred to as exonucleases) to the gene editing system, and finally found that T7 exonuclease (also known as T7 phage exonuclease) Enzyme) can produce long fragments of single-stranded DNA at the DSB in the cell. The long fragments of single-stranded DNA are conducive to the repair of the HDR pathway, but cannot be repaired by the NHEJ pathway, thereby reducing the repair efficiency of the NHEJ pathway and improving the HDR pathway. Repair efficiency.

According to an embodiment of the present invention, the T7 exonuclease has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology therewith. The inventors have found through a large number of experiments that using an exonuclease homologous to T7 exonuclease can reduce the repair efficiency of the NHEJ pathway and improve the repair efficiency of the HDR pathway.

The amino acid sequence of T7 exonuclease is shown below (SEQ ID NO: 1):

gene editing system

In yet another aspect of the present invention, the present invention provides a gene editing system. According to an embodiment of the present invention, the gene editing system comprises: a site-specific nuclease, a 5'→3' exonuclease and a donor DNA; wherein, the 5'→3' exonuclease and the Site-specific nucleases are non-fused.

The inventors have found through experiments that after the above-mentioned site-specific nuclease, 5'→3' exonuclease and donor DNA are delivered into the cell, the site-specific nuclease produces a DSB in the cell, and then the 5' →The 3'exonuclease functions in the cell line to generate long fragments of single-stranded DNA, thereby changing the HDR/NHEJ ratio in the editing results, reducing the repair efficiency of the NHEJ pathway, and improving the repair efficiency of the HDR pathway.

It should be noted that the delivery method of the present invention is not limited, and can be selected according to specific needs, specifically, plasmid overexpression system: including plasmid chemical transfection, plasmid electroporation, etc.; viral expression system: adeno-associated virus (AAV) infection, lentivirus infection, etc.; other expression systems: including protein/RNA complex electroporation, mRMA electroporation, mRMA chemical transfection, etc.

As used herein, the term "donor DNA" includes a nucleic acid sequence to be inserted into cellular DNA such as genomic DNA, mitochondrial DNA or viral DNA. The donor nucleic acid sequence can be expressed by the cell. The donor nucleic acid can be exogenous, foreign to the cell or non-naturally occurring within the cell.

The donor DNA is associated with a sequence that can be bound by the DNA binding domain. For example, the donor DNA may be adjacent to the consensus sequence of the DNA binding domain, or may be present on the same vector as the consensus sequence, or may be present on the same polynucleotide as the consensus sequence.

According to an embodiment of the present invention, the added amount of the site-specific nuclease is 2-50 ng. The inventor obtained the above-mentioned optimal addition amount through a large number of experiments, thus, the repair efficiency of the improved HDR channel can be further filled.

According to an embodiment of the present invention, the added amount of the 5'→3' exonuclease is 40-120ng. The inventor obtained the above-mentioned optimal addition amount through a large number of experiments, thus, the repair efficiency of the improved HDR channel can be further filled. Moreover, the inventors found that an excessively high concentration of 5'→3' exonuclease would disrupt the occurrence of both NHEJ and HDR editing.

According to an embodiment of the present invention, the added amount of the donor DNA is 0.2-100 ng. The inventor obtained the above-mentioned optimal addition amount through a large number of experiments, thus, the repair efficiency of the improved HDR channel can be further filled.

According to an embodiment of the present invention, the 5'→3' exonuclease is T7 exonuclease. Therefore, compared with other 5'→3' exonucleases, T7 exonuclease can produce long fragments of single-stranded DNA at DSBs in cells, and the appearance of this long fragment of single-stranded DNA is conducive to the repair of HDR pathway, Inhibit the repair of NHEJ pathway, thereby reducing the NHEJ pathway, while improving the repair efficiency of the HDR pathway.

According to an embodiment of the present invention, the T7 exonuclease has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology therewith. The inventors have found through a large number of experiments that using an exonuclease homologous to T7 exonuclease can reduce the repair efficiency of the NHEJ pathway and improve the repair efficiency of the HDR pathway.

According to an embodiment of the present invention, the site-specific nuclease is selected from endonucleases, specifically, the site-specific nuclease can be selected from clustered regularly interspaced short palindromic repeats, transcription activation-like effectors At least one of nuclease, zinc finger nuclease, homing endonuclease, and restriction endonuclease. According to an embodiment of the present invention, in the application of the gene editing system, the above-mentioned site-specific nucleases can be used to produce DSBs according to requirements, and the specific types are not limited.

It should be noted that, the specific nucleases for the above sites can be selected from endonucleases such as clustered regularly interspaced short palindromic repeats, transcription activator-like effector nucleases, zinc finger nucleases, and homing endonucleases. At least one of them may also be a nuclease modified on the basis of any of the above-mentioned site-specific nucleases (for example, introducing a point mutation), and the specific type is not limited.

According to an embodiment of the present invention, the gene editing system further comprises: gRNA. If the gene editing system is a CRISPR system, gRNA and cluster regularly interspaced short palindromic repeats (CRISPR/Cas9, hereinafter referred to as Cas9) are used to jointly produce DSB.

According to an embodiment of the present invention, the gRNA includes at least one selected from crRNA/tracrRNA, sgRNA, and pegRNA. According to an embodiment of the present invention, in the gene editing system, as long as the guide RNA is capable of forming a complex with the site-specific nuclease and can target the complex to the target due to certain complementarity with the target sequence The sequence is enough, and at least one of the above-mentioned guide RNAs (the guide RNAs in the present invention include traditional guide RNAs and guide RNAs optimized and improved on traditional guide RNA sequences) can be used, wherein the specific type is not limited.

According to an embodiment of the present invention, the gene editing system further includes: a recruitment system. The inventors found through experiments that by adopting the recruitment system, a large amount of 5'→3' exonuclease can be enriched, which can further improve the repair efficiency of the HDR pathway and reduce the repair efficiency of the NHEJ pathway.

It should be noted that as long as the recruitment system in the present invention can enrich 5'→3' exonuclease in a large amount, it can be a recruitment system for protein-small molecule interaction. Recruitment system, nucleic acid-protein interaction recruitment system, protein-protein interaction recruitment system, for example, MS2/MCP system, PP7-PCP system, Suntag system, etc. can be selected as the recruitment system, and the specific method is not limited.

According to an embodiment of the present invention, if the recruitment system is the MS2/MCP system, the gRNA is gRNA-MS2, and the 5'→3' exonuclease is an MCP-exonuclease fusion protein. In this way, the 5'→3' exonuclease can be enriched in large quantities, and the repair efficiency of the HDR pathway can be further improved.

According to an embodiment of the present invention, the donor DNA is a linear double-stranded DNA modified with a phosphorothioate bond and/or a circular double-stranded DNA in the form of HMEJ.

The inventors found through experiments that by introducing end protection means to protect the end of the donor DNA as a replication template for the HDR pathway, the donor DNA can be stably present in the cell before editing begins, further improving the efficiency of gene editing, and making the editing efficiency Significantly increased. According to an embodiment of the present invention, compared with unmodified linear double-stranded DNA, the above-mentioned donor DNA used in the present invention has higher HDR pathway repair efficiency and HDR/NHEJ ratio.

According to an embodiment of the present invention, the number of modifications of the phosphorothiodiester bonds on the donor DNA is greater than 2, preferably 4-10. As a result, the repair efficiency of the HDR pathway and the HDR/NHEJ ratio can be further improved.

According to an embodiment of the present invention, the technical route of the gene editing system includes homologous recombination mediated by an oligonucleotide editing template, single base editing, guided editing, and homologous recombination mediated by a double-stranded long-chain nucleic acid editing template One of.

Those skilled in the art can understand that the features and advantages described above for the use of the 5'→3' exonuclease in the gene editing system are also applicable to the gene editing system, and will not be repeated here.

Methods for gene editing cells

In another aspect of the present invention, the present invention provides a method for gene editing of cells. According to an embodiment of the present invention, the method includes: introducing the above-mentioned gene editing system into cells. The inventors found that introducing the above-mentioned gene editing system into cells can improve the HDR editing efficiency and the HDR/NHEJ ratio in cells.

According to an embodiment of the present invention, the cells are animal cells, preferably mammalian cells. The inventors have found through a large number of experiments that the repair efficiency of the HDR pathway can be improved by adding 5'→3' exonuclease, which is suitable for the gene editing system of animal cells, especially for mammalian cells.

According to an embodiment of the present invention, the cells are terminally differentiated primary cells. The inventors found through experiments that T7 exonuclease, as an exogenous protein, has the characteristics of not being clearly regulated by the cell cycle, so that the end-cutting process can occur in various stages of the cell cycle, and can induce the HDR pathway in the terminal differentiation primary Cells such as non-dividing cells occur.

Those skilled in the art can understand that the features and advantages described above for the use of 5'→3' exonuclease in the gene editing system and the gene editing system are also applicable to the method for gene editing of cells, I won't repeat them here.

The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Example 1: Comparison of the repair efficiency of different 5'→3' exonucleases in the gene editing system for the HDR pathway

The gene editing system of this embodiment includes the following steps:

1. Construction of expression vector and donor DNA

sgRNA sequence design can be done through design websites such as benchling. Donor DNA is recommended to use 700bp homology arms, constructed as HMEJ donor format. Other homology arm lengths and donor DNA forms can also be used for editing purposes.

The DSB is produced by the CRISPR/Cas9 system, and the exonuclease is enriched by the MS2 recruitment system, wherein the exonuclease is T7 bacteriophage exonuclease, T5 phage exonuclease (the amino acid sequence is shown in SEQ ID NO: 2 ), lambda bacteriophage exonuclease (as shown in SEQ ID NO: 3), Escherichia coli exonuclease DCLRE1B (as shown in SEQ ID NO: 4), Escherichia coli exonuclease RecE (amino acid The sequence is shown in SEQ ID NO: 5) or human exonuclease EXO1 (amino acid sequence is shown in SEQ ID NO: 6), and the amino acid sequence of T7 bacteriophage exonuclease is shown in SEQ ID NO: 1.

Amino acid sequence of T5 bacteriophage exonuclease:

Amino acid sequence of lambda bacteriophage exonuclease:

Amino acid sequence of Escherichia coli exonuclease DCLRE1B:

Amino acid sequence of Escherichia coli exonuclease RecE:

Amino acid sequence of human exonuclease EXO1:

2. Delivery of editing system to target cells

Through the plasmid overexpression system, the specific steps are as follows:

1) Inoculate 293T cells in a 96-well cell culture plate and culture overnight at 37° C. in a 5% carbon dioxide environment.

2) The expression vector containing the gene encoding Cas9 protein and sgRNA-MS2 (abbreviated as Cas9/sgRNA expression vector or Cas9/sgRNA) and the expression vector containing the gene encoding MCP-exonuclease fusion protein were transferred by transient transfection (referred to as MCP-exonuclease fusion protein expression vector or MCP-exonuclease Dicer fusion protein) was introduced into the cell line together with donor DNA, wherein the amount of transfected plasmid added was: Cas9/sgRNA 50ng, MCP-exonuclease fusion protein 100ng, and donor DNA 50ng. Unless otherwise specified, the donor DNA used was HMEJ donor, the total amount of plasmid was 200ng, and the transfection reagent was 0.75 μL. Transfection was performed by PEI (1 mg/mL) when the cell density reached 80%-90%. Or transfect by lipo2000 when the cell density reaches 50%-60%.

Among them, the Cas9/sgRNA expression vector and the MCP-exonuclease fusion protein expression vector can be constructed using conventional techniques in the art, for example, see "Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6,343 -345, doi:10.1038/nmeth.1318(2009)" and "Potapov, V. et al. Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4DNA Ligase and Application to DNA Assembly. ACS Synth Biol 7, 2665-2674, do i :10.1021/acssynbio.8b00333 (2018)".

The amino acid sequence of the Cas9 protein is:

The amino acid sequence of sgRNA-MS2 is as follows, N represents any one of A, T, C, and G, uppercase letters represent the spacer region, designed according to the editing site, and lowercase letters represent the backbone region:

The amino acid sequence of MCP is as follows, the C-terminal of the MCP protein fragment is connected to the N-terminal of the exonuclease fusion protein fragment:

The nucleotide sequences corresponding to the protein fragments in this example are based on their amino acid sequences using conventional methods or conventional software.

3) Continue culturing the transfected cells for 48 hours at 37° C. in a 5% carbon dioxide environment.

4) According to the purpose of the experiment, carry out downstream detection and analysis.

3. Detect the editing results and apply them downstream

Insert the TLR reporter system (as shown in Figure 1) into the cell genome for detection, and the specific steps are as follows:

1) Aspirate the cell culture medium in the well, slowly add 50 μL DPBS solution to wash the cells.

2) The DPBS solution was sucked off, and 20 μL of 0.25% trypsin was added to digest the cells for 1 min.

3) Add 80 μL of complete medium and resuspend the cells by pipetting.

4) Transfer the cells to a round-bottom 96-well cell culture plate, centrifuge at 100 g for 3 min, and discard the supernatant.

5) Use 100 μL of serum-free medium to resuspend the cells.

6) Collect data using a flow cytometer. The mKate fluorescence signal corresponds to the repair of the NHEJ pathway, and the EGFP fluorescence signal corresponds to the repair of the HDR pathway.

4. Results

The repair efficiency of the HDR pathway is shown in Figure 2, where the abscissa indicates the species source of the exonuclease, and all the exonucleases form fusion proteins with MS2, from left to right: no exonuclease negative control, T7 phage Exonuclease, T5 bacteriophage exonuclease, lambda bacteriophage exonuclease, E. coli exonuclease DCLRE1B, E. coli exonuclease RecE and human exonuclease EXO1 active domain; ordinate indicates repair efficiency.

The results showed that exonucleases from each source were functional in cell lines, altering the HDR/NHEJ ratio in the edited results. Among them, T7 exonuclease most significantly reduced the repair efficiency of the NHEJ pathway, while improving the repair efficiency of the HDR pathway.

Example 2: Comparison of the repair efficiency of different donor DNAs in the gene editing system for the HDR pathway

The difference between the gene editing system in this example and Example 1 is that the exonuclease is T7 exonuclease. In this example, different donor DNAs are selected for experiments, that is, unmodified linear double-stranded DNA, sulfo Phosphodiester bond (PS)-modified linear double-stranded DNA (nPS refers to the 5' end of linear double-stranded DNA modified by n consecutive phosphorothioate bonds) and circular double-stranded DNA in the form of HMEJ were used as donors.

As shown in Figure 3, where the abscissa indicates the type of donor DNA, PCR: linear double-stranded DNA, nPS: linear double-stranded DNA with n PS modifications at the 5' end, HMEJ: circular double-stranded DNA in the form of HMEJ; vertical Coordinates indicate repair efficiency.

The results showed that after the introduction of end protection means, the HDR editing efficiency was significantly improved, and the HMEJ donor had the highest HDR editing efficiency and HDR/NHEJ ratio.

Example 3: Research on the editing efficiency of sites of different lengths near the cleavage site

In this example, the HMEJ double-stranded DNA donor is used to introduce consecutive three-base mutations at different positions within 100 bp from the cutting site (nBP refers to the distance between the introduced mutation position and the Cas9 cutting site by n base pairs). The gene editing efficiency was characterized by next-generation sequencing analysis, and the rest of the steps were the same as in Example 1.

Among them, the specific steps of next-generation sequencing analysis to characterize gene editing efficiency are as follows:

1. Use 0.25% trypsin to digest and collect the cells.

2. Centrifuge at 500g to pellet the cells and remove the supernatant.

3. If the transiently transfected expression vector has a fluorescent label, flow sorting technology can be used to screen out the cells successfully introduced into the editing system according to the fluorescent label, and then proceed to the downstream steps. This method is generally applicable to cell lines with low transfection efficiency.

4. Use Quick extract (Epicentre) reagent to resuspend the cells, incubate at 65°C for 15 minutes, at 68°C for 15 minutes, and at 95°C for 10 minutes to obtain the genome fragment solution.

5. Use a primer outside the homology arm region near the editing site to perform PCR amplification of the genomic sequence near the editing site. Amplification was performed for 35 cycles to eliminate interference from donor DNA.

6. Dilute the product in step 5 with ultrapure water at a ratio of 1:10, take 1 μL as a template, and use a pair of primers near the editing site to build a library according to the requirements of next-generation sequencing. This step does not exceed 20 cycles of amplification . This step requires the full length of the PCR product to be 150bp-200bp (excluding the part of the next-generation sequencing adapter).

7. Use CRISPResso2 (specific references Li X, Wang Y, Liu Y, et al. Base editing with a Cpf1-cytidine deaminase fusion [J]. Nat Biotechnol, 2018, 36(4): 324-327) to sequence the results Analyze and count the proportion of NHEJ pathway repair and HDR pathway repair.

As shown in Figure 4, where the abscissa represents the distance between the mutation site and the cleavage site, and the ordinate represents the repair efficiency. The results show that the editing site closer to the cutting site has higher editing efficiency, and effective editing can still be completed at a distance of at least 100 bp from the cutting site.

Example 4: Research on the Editing Efficiency of Different Sites of the Genome

In this example, the HMEJ double-stranded DNA donor was used to edit the four endogenous sites of HEK3, DNMT1, RNF2, and VEGFA in the cell genome respectively, and the gene editing efficiency was analyzed and characterized by next-generation sequencing (refer to Example 3), and other steps were the same Example 1. Wherein, the amount of plasmid transfection in this embodiment is 20ng of Cas9/sgRNA, 80ng of T7 exonuclease, and 100ng of donor DNA.

As shown in Figure 5, where the abscissa indicates the name of the gene where the editing site is located or the name of the editing site, and the ordinate indicates the repair efficiency, T7+ indicates the addition of T7 exonuclease during the editing process, and T7- indicates that T7 is not added during the editing process exonuclease. The results showed that different sites in the genome had differences in editing efficiency and repair pathway selection, but high-efficiency HDR pathway editing could be completed at all sites.

Example 5: Protein labeling effect of gene editing system in cell lines

In this example, the HMEJ double-stranded DNA donor was used to knock in FlAsH short peptide tags at the C-terminus of the NPM1 gene site (Figure 6) and the N-terminus of the FUS gene site (Figure 7) of the genome (specific references Griffin B A, Adams S R, Tsien R Y.Specific covalent labeling of recombinant protein molecules inside live cells[J].Science,1998,281(5374):269-72 and Thorn K S,Naber N,Matuska M,et al.A novel method of affinity-purifying proteins using a bis-arsenical fluorescein[J]. Protein Sci, 2000,9(2):213-7), to observe the cellular localization of the target protein in living cells. Wherein, the amount of plasmid transfection in this embodiment is Cas9 20ng, T7 exonuclease 80ng, donor DNA 100ng.

The results show that this editing tool can introduce fluorescent tags very efficiently, thereby displaying the cellular localization of proteins in living cells (NPM1 localization: nucleolus, FUS localization: the area outside the nucleolus; left: fluorescently labeled endogenous proteins, right Figure: hoechst marks chromatin), which can facilitate the functional study of the endogenous expression level of the protein.

Example 6: Research on Editing Efficiency of T7 Exonuclease in PE Editing System

This example improves the PE editing system, restores the Cas9 nickase (Cas9n) mutation to wild-type Cas9, extends the primer binding region and uses it in conjunction with T7 exonuclease, and analyzes it by next-generation sequencing (reference example 3) To characterize gene editing efficiency, use 50ng of expression plasmids containing genes encoding Cas9-MMLV fusion protein/pegRNA during transfection, 100ng of expression plasmids containing genes encoding T7 exonuclease as the experimental group; 50ng containing expression plasmids encoding Cas9n-MMLV fusion proteins The expression plasmid of the gene of/pegRNA, 100ng pUC19 plasmid is used as PE system control, and other steps are with embodiment 1.

As shown in Figures 8 and 9, the results showed that the improved PE-T7 editing system (ie, px330-mmlv/t7) showed significantly higher gene editing efficiency than the PE system for multiple genomic sites tested. (cutting in the figure refers to the total editing efficiency caused at the genomic site, and ins/del refers to the efficiency of introducing insertion/deletion mutations as expected).

Example 7: Comparison with the presence or absence of the recruitment system

The difference between the gene editing system in this example and Example 1 is that the exonuclease is T7 exonuclease, and the HDR pathway with/without MS2 recruitment system was compared by using sgRNA without MS2-loop structure as a control Repair efficiency.

As shown in Figure 10, the results showed that after the introduction of the MS2 recruitment system, T7 exonuclease inhibited the NHEJ pathway and enhanced the function of the HDR pathway.

Example 8: Study of Single-Stranded DNA Manufactured at Intracellular DSBs by T7 Exonuclease

Donor DNA was not used in this example, only Cas9/sgRNA and T7 exonuclease were introduced as the experimental group (i.e., T7+), and samples were taken at different time points after transfection, and single samples were analyzed by enzyme digestion-fluorescent quantitative PCR method. stranded DNA is detected, and the control uses MCP protein instead of T7 exonuclease (being T7-) (in this embodiment, a 6-well cell culture plate is used, and during transfection, the contents of Cas9/sgRNA and T7 exonuclease are respectively 500ng, 1500ng, PEI dosage is 12μL).

As shown in FIG. 11 , the results showed that within a period of time after transfection, the total amount of single-stranded DNA near the DSB of the sample added with T7 exonuclease was significantly higher than that of the control group.

Example 9: Research on editing efficiency of T7 exonuclease in different cell lines

In this example, gene editing was performed on various cell lines using the HMEJ double-stranded DNA donor. In the experiment, the cells that were successfully introduced into the editing tool were screened by flow cytometry technology, and the gene editing efficiency was analyzed and characterized by next-generation sequencing (refer to Example 3). Transfection, the amount of transfection reagent is 1.5μL, the contents of Cas9/sgRNA, T7 exonuclease and donor DNA during transfection are: HCT116, 50ng, 250ng, 200ng; U2OS: 250ng, 350ng, 300ng; Hela, 50ng, 250ng, 200ng; mESC: 200ng, 200ng, 150ng) Other steps are the same as in Example 1, and the test results are shown in Figures 12-18. The results show that in various cell lines, the introduction of T7 exonuclease can inhibit the repair of the NHEJ pathway and significantly improve the repair efficiency of the HDR pathway. Therefore, the efficient editing performance of the gene editing system of the present invention does not depend on the cell system.

Example 10: Research on Editing Efficiency of T7 Exonuclease in Primary Rat Neurons

In this example, the HMEJ double-stranded DNA donor was used to express the cortical nerve cells of E18 rat embryos by electroporation using a Lonza-4D electroporation instrument. For vector delivery, use 20μL electroporation system, the contents of Cas9/sgRNA, T7 exonuclease and donor DNA are: 500ng, 1000ng, 500ng respectively (refer to https://bioscience.lonza.com/lonza_bs/DE for specific operation methods /en/download/product/asset/21243), the efficiency of gene editing was characterized by next-generation sequencing analysis, other steps were the same as in Example 1, and the analysis results are shown in Figure 19. The results show that the editing tool can complete effective gene editing in rat primary nerve cells. Therefore, the gene editing system of the present invention can realize HDR editing in terminally differentiated primary cells.

Example 11: Effect of specific endonuclease transfection concentration on editing results

In this example, HMEJ double-stranded DNA donors are used to edit different sites in the cell genome, and the gene editing efficiency is analyzed and characterized by next-generation sequencing (refer to Example 3). Other steps are the same as in Example 1. The analysis results are shown in Figure 20- twenty four. And tested the Cas9/sgRNA expression plasmids with different transfection amounts in editing, and used the pUC19 plasmid to complete the total transfection amount. The results showed that a higher HDR/NHEJ ratio could be obtained when the endonuclease concentration was lower, and a higher HDR repair efficiency could be obtained when the endonuclease concentration was higher.

Example 12: Effect of T7 exonuclease transfection concentration on editing results

In this example, HMEJ double-stranded DNA donors were used to edit different sites in the cell genome, and the gene editing efficiency was analyzed and characterized by next-generation sequencing (refer to Example 3). The content of Cas9/sgRNA was 20ng, and other steps were the same as in Example 1. , the analysis results are shown in Figure 25-30. And tested the use of T7 exonuclease expression plasmids with different transfection amounts in editing, and used the MCP protein expression plasmid to complete the total amount of transfection. The results showed that as the concentration of T7 exonuclease increased, the efficiency of NHEJ showed a downward trend, and the efficiency of HDR showed a trend of first increasing and then decreasing.

Example 13: Effect of Donor DNA Transfection Concentration on Editing Results

In this example, HMEJ double-stranded DNA donors were used to edit different sites in the cell genome, and the gene editing efficiency was analyzed and characterized by next-generation sequencing (reference example 3). The content of Cas9/sgRNA was 20ng, and the content of T7 exonuclease 80ng, the other steps are the same as in Example 1, and the analysis results are shown in Figures 30-34. And tested the use of different transfection amounts of donor DNA plasmids in editing, using the pUC19 plasmid to complete the total amount of transfection. The results showed that as the concentration of donor DNA increased, the efficiency of NHEJ pathway decreased and that of HDR pathway increased.

Example 14: Dual fluorescent knock-in editing of NPM1

This embodiment is an extended application of Embodiment 5. In the experiment, the donor DNA with the split-sfGFP tag introduced at the C-terminal of the NPM1 protein (that is, NPM1 ^WT ) and the donor DNA with the pathogenic +4 mutation and the FlAsH short peptide tag introduced at the C-terminal of the NPM1 protein (that is, the NPM1 ^mut ). The Cas9/sgRNA content is 50ng, the T7 exonuclease content is 80ng, and the donor DNA is 100ng. Other steps are the same as in Example 5. Fluorescence-labeled double-positive cells were sorted by flow cytometry, and the fluorescence was observed by confocal microscopy (Figure 37, (a): DAPI; (b): NPM1 ^WT ; (c): NPM1 ^mut ) . The co-localization of the introduced fluorescent label and NPM1 protein wild-type/mutant immunofluorescence was observed separately (Figure 38, (a) NPM1 ^WT DAPI (b) NPM1 ^WT fluorescent label (c) NPM1 ^WT immunofluorescence staining (d) NPM1 ^mut DAPI (e) NPM1 ^mut fluorescent label (f) NPM1 ^mut immunofluorescent staining. The results show that this method can introduce different mutations to different alleles of the same gene in a cell at the same time, and efficiently create double knock-in hybrid cells .

Example 15: Research on FUS protein disease-associated mutants by means of gene editing

This embodiment is an extended application of Embodiment 5. The cell line in which the FlAsH tag was knocked in the FUS protein obtained in Example 5 was used in the experiment. In this experiment, a currently discovered disease-associated point mutation was introduced into the FUS protein by a gene editing method (this example takes a point mutation in the C-terminal NLS region as an example), and the behavioral changes of the mutant protein in cells were observed by a fluorescence microscope. The Cas9/sgRNA content is 50ng, the T7 exonuclease content is 80ng, and the donor DNA is 50ng each. Other steps are the same as in Example 5. The cells were stimulated with 500 μM sodium arsenite for 1 h, and by observing the ratio of FUS protein entering the stress granules (SGs) in cells introduced with different point mutations, a group of mutants that caused FUS to localize to SGs were screened (Figure 39) , the subcellular localization of mutant and wild-type FUS was significantly different (Fig. 40, (a) P525L mutant; (b) wild-type). Then, the screening results were verified by single-cell culture and immunofluorescence staining to verify the corresponding relationship between the mutation and the phenotype, and the results are shown in Figure 41 (Figure 41, (a) WT DAPI; (b) WT FUS (c); Q519X DAPI; (d) ); Q519X FUS; (e) P525L DAPI; (f) P525L FUS). The results show that this method can be used for high-throughput screening and preliminary functional identification of point mutations in various proteins.

Example 16: Study on the effect of polyQ structure length of ATXN2 protein on protein function by means of gene editing

The ATXN2 protein contains a glutamine repeat sequence domain (polyQ). The length of polyQ in the wild-type protein is 22-23. The abnormal extension of its sequence is related to the pathogenesis of various diseases. In this example, the FlAsH fluorescent label was introduced into the ATXN2 protein by the method of Example 5, and the mutation was introduced into the polyQ region of the ATXN2 protein by the method of Example 15, and ATXN2 gene editing cell lines with different polyQ lengths were constructed (nQ refers to the gene editing cell ATXN2 in the system The protein polyQ domain contains n consecutive glutamines). We used Western-blotting to detect the molecular weight change of the ATXN2 degradation band, and verified the editing results by first-generation sequencing (Figure 42, (a) Western-blotting 70kD-80kD ATXN2 band; (b) polyQ sequence generation sequencing of 44Q cell line result). Based on this, the effect of polyQ length on the behavior of ATXN2 in the process of cell stress and recovery was explored and summarized by Western-blotting (Figure 43, left: WT; middle: 30Q; right: 44Q; each group was : before stress, recovery 0h after stress, recovery 2h after stress, recovery 6h after stress). The results show that this method can avoid the introduction of concentration changes when studying mutant proteins, which will interfere with the experimental results.

Example 17: Testing the length range of mutations introduced by gene editing tools

In this example, the donor DNA with insertion mutation or substitution mutation was used, and the rest of the experimental methods were the same as in Example 3. As shown in Figures 35 (insertion mutation) and 36 (replacement mutation), the abscissa represents the length of the insertion/replacement mutation introduced, and the ordinate represents the repair efficiency. The results show that the editing tool can effectively edit at least 200bp DNA fragments.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, those skilled in the art can make the above-mentioned The embodiments are subject to changes, modifications, substitutions and variations.

Claims (24)

1. A use of a 5'→3' exonuclease in a gene editing system, characterized in that the 5'→3' exonuclease is non-specific to the site-specific nuclease in the gene editing system fusion state.
2. The use according to claim 1, characterized in that the cells used in the gene editing system are animal cells.
3. The use according to claim 2, characterized in that the cells used in the gene editing system are mammalian cells.
4. purposes according to claim 1, is characterized in that, described 5 ' → 3 ' exonuclease is T7 exonuclease.
5. The use according to claim 4, wherein the T7 exonuclease has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology therewith.
6. A gene editing system, characterized in that it comprises:

   Site-specific nucleases, 5'→3' exonucleases, and donor DNA;

   Wherein, the 5'→3' exonuclease is in a non-fused state with the site-specific nuclease.
7. The gene editing system according to claim 6, wherein the 5'→3' exonuclease is T7 exonuclease.
8. The gene editing system according to claim 7, wherein the T7 exonuclease has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology therewith.
9. The gene editing system according to any one of claims 6 to 8, wherein the site-specific nuclease is selected from the group consisting of clustered regularly interspaced short palindromic repeats, transcriptional activation-like effector nucleases, and zinc finger nucleic acids At least one of enzymes, homing endonucleases, and restriction endonucleases.
10. The gene editing system according to any one of claims 6-8, wherein the added amount of the site-specific nuclease is 2-50 ng.
11. The gene editing system according to any one of claims 6-8, wherein the added amount of the 5'→3' exonuclease is 40-120ng.
12. The gene editing system according to any one of claims 6-8, characterized in that the added amount of the donor DNA is 0.2-100 ng.
13. The gene editing system according to claim 9, wherein the gene editing system further comprises: gRNA.
14. The gene editing system according to claim 13, wherein the gRNA comprises at least one selected from sgRNA, pegRNA and crRNA/tracrRNA.
15. The gene editing system according to claim 14, wherein the gene editing system further comprises: a recruitment system.
16. The gene editing system according to claim 15, wherein if the recruitment system is the MS2/MCP system, the gRNA is gRNA-MS2 or pegRNA-MS2, and the 5'→3' exonuclease is MCP-exonuclease fusion protein.
17. The gene editing system according to any one of claims 6 to 8, wherein the donor DNA is a linear double-stranded DNA modified with a phosphorothioate bond and/or a circular double-stranded DNA in the form of HMEJ .
18. The gene editing system according to claim 17, wherein the number of modifications of the phosphorothioate bonds on the donor DNA is greater than 2.
19. The gene editing system according to claim 18, wherein the number of modifications of the phosphorothioate bonds on the donor DNA is 4-10.
20. The gene editing system according to any one of claims 6 to 8, wherein the technical route of the gene editing system includes homologous recombination mediated by oligonucleotide editing templates, single base editing, guided editing and A type of homologous recombination mediated by double-stranded long-chain nucleic acid editing templates.
21. A method for genetically editing cells, comprising:

    Introducing the gene editing system according to any one of claims 6-20 into cells.
22. The method of claim 21, wherein the cells are animal cells.
23. The method of claim 22, wherein the cells are mammalian cells.
24. The method according to any one of claims 21-23, wherein the cells are terminally differentiated primary cells.