CN104450785A - Genome editing method using attachment carrier for encoding targeted endonuclease and kit - Google Patents

Abstract

The invention belongs to the technical field of biological genetic engineering, and specifically relates to a genome editing method and a kit using an attachment carrier encoding a targeting endonuclease. The present invention uses the attachment carrier to express the targeted endonuclease, the attachment carrier can realize non-integrated stable gene expression, and express the screening gene at the same time; the cells without plasmid can be removed through drug screening; the drug can be removed after the editing is completed , the cells will gradually lose the episomal vector, so that the edited cell line without the foreign gene is obtained. The method prolongs the editing time and improves the gene editing efficiency. The invention also provides kits for editing specific chromosomal sequences in cells. The invention is applicable to genome editing of various eukaryotic cells.

Description

Methods and kits for genome editing using attachment vectors encoding targeting endonucleases

field of invention

The invention belongs to the technical field of genetic engineering, and in particular relates to a genome editing method and a kit.

Background technique

Genome editing is a genetic manipulation technology that can modify DNA sequences at the genome level. The principle of this technology is to construct an artificial endonuclease to cut DNA at a predetermined genomic position, and the cut DNA will undergo mutations during repair by the DNA repair system in the cell, thereby achieving the purpose of targeted genome modification. The DNA repair system mainly repairs DNA double-strand breaks through two ways, namely, non-homologous end joining and homologous recombination. Through these two repair pathways, genome editing technology can achieve three genome modification goals, namely, gene knockout, introduction of specific mutations, and site-directed transgenesis. 1) Gene knockout: If you want to lose the function of a certain gene, you can generate a DNA double-strand break on the gene. During the repair of non-homologous end junctions, DNA insertions or deletions often occur, resulting in frameshift mutations. In order to achieve gene knockout; 2) Introduction of specific mutations: If you want to introduce a specific mutation into the genome, you need to achieve it through homologous recombination. At this time, you need to provide a homologous template containing specific mutations. Under normal circumstances, the efficiency of homologous recombination is very low, and the generation of double-strand breaks at this site will greatly improve the recombination efficiency, thereby realizing the introduction of specific mutations; 3) Site-directed transgene: the same principle as the introduction of specific mutations, A transgene is added in the middle of the template, and this transgene will be copied into the genome during the double-strand break repair process, thereby realizing site-specific transgenesis.

Currently popular genome editing technologies include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and regularly clustered interspaced short palindromic repeat ( Clustered regularly interspaced short palindromic repeats, CRISPR/Cas9).

Zinc finger enzymes (ZFNs) consist of a DNA recognition domain and a nonspecific endonuclease. The DNA recognition domain is composed of 3-4 Cys2-His2 zinc finger proteins in series, and each zinc finger protein recognizes and binds a specific triplet base. Multiple zinc finger proteins are connected in series to form a zinc finger protein group to recognize a specific base sequence (9-12 bp), which has strong specificity. The non-specific endonuclease linked to the zinc finger protein group comes from the C-terminal DNA cleavage domain of FokI, which has enzymatic cleavage activity only in the dimer state. Each FokI monomer is connected with a zinc finger protein group to form a ZFN, which recognizes a specific site. When the two recognition sites are at an appropriate distance (6-8 bp), the two monomer ZFNs interact to produce restriction enzymes Function. In order to achieve the purpose of site-specific shearing of DNA.

TALEN technology is a molecular biology tool developed after ZFN technology. Boch and Moscou et al. found that the amino acid sequence of the nucleic acid binding domain of the TALE protein from the plant bacterium Xanthomonassp. has a fixed correspondence with the nucleic acid sequence of its target site. The nucleic acid recognition unit of TALE is to repeat 34 constant amino acid sequences, and the double-linked amino acids at positions 12 and 13 have a constant corresponding relationship with A, G, C, and T, that is, NG recognizes T, HD recognizes C, NI recognizes A, NN recognizes G. These four TAL modules can be assembled to specifically bind any DNA sequence. Cermak et al. used TALE modules to replace the DNA recognition domains in ZFNs, and assembled them into a new genome editing tool, TALEN (Tal effector nuclease), to achieve the purpose of targeted genome editing.

CRISPR/Cas9 is a newly emerged technology for targeted gene editing by RNA-guided Cas9 nuclease. CRISPR/Cas9 is an adaptive immune defense mechanism evolved by bacteria and archaea in response to constant attacks by viruses and plasmids. In this system, crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a double-stranded RNA, and the tracrRNA/crRNA binary complex guides the Cas9 protein to cut at the target site of the crRNA guide sequence double-stranded DNA. During the genome editing process, tracrRNA and crRNA can be fused into one RNA (sgRNA, referred to as guide RNA in the present invention), and the expression can also play the role of targeted cutting. The advantage of CRISPR/Cas9 is that it is easy to operate and has high efficiency on the genome. When it is necessary to edit a certain target site, it only needs to express the corresponding guide RNA, and there is no need to modify the Cas9 nuclease. However, recent studies have shown that there is a certain off-target shearing phenomenon in the CRISPR/Cas9 system, which hinders the widespread application of this technology. In order to overcome the phenomenon of off-target shearing, Feng Zhang, an assistant professor of brain and cognitive science at the Massachusetts Institute of Technology, a core member of the McGovern Brain Institute and the Broad Institute, and others used a mutant endonuclease Cas9n that only produces a single nick on DNA. (Cas9-D10A) and two guide RNAs with closer recognition sites are expressed in cells at the same time, and Cas9n cuts the two strands of DNA respectively to generate DNA breaks, which improves the specificity.

Episomal vectors are a class of DNA plasmids that are episomal and can replicate in eukaryotic cells. Currently popular episomal vectors include Epstein-Barr virus (EBV), BK virus (BKV), bovine papilloma virus 1 (BPV-1), simian virus 40 (SV40) derived plasmid vectors or a type of S/MAR (Scaffold/Matrix Attachment Region, S/MAR)-mediated non-viral attachment carrier.

EBNA1/oriP is an important gene element related to the replication of EBV and maintaining the episomality of the viral genome. EBNA1 is the gene of EBV nuclear antigen L, which encodes a phosphorylated and protein. The C-terminus of the protein is a dimerized DNA-binding region, and EBNA1 homologously dimerizes and binds to a specific sequence on DNA. OriP contains double symmetry (dyad symmetry DS) and clustered repeat sequence (the family of repeats, FR). These sequences are included in the EBNA1 binding site. When EBNA1 is combined with these sequences, the viral genome exists in a stable episomal form, and the viral genome replicates in a host-dependent manner. In view of the characteristics of EBNA1/oriP, Yale introduced these two genetic elements into plasmids for the first time in 1985. EBNA1/oriP plasmids are not integrated after transfection into target cells, and can exist stably for a long time.

BKV belongs to the papovaviridae family, polyomavirinae subfamily, and was first isolated in the urine of a kidney transplant patient in 1971. BKV can efficiently infect mammalian cells and keep viral DNA episomal outside the host chromosome. According to this characteristic, the BKV large T antigen and the replication initiation sequence can be made into an attachment plasmid for stably expressing foreign genes. As used herein, the term "foreign" refers to any sequence not normally found at this chromosomal location. For example, an exogenous sequence can be a "gene" from another organism, an additional copy of a "gene" from the same organism, an artificial sequence, a sequence encoding a reporter, and the like. Papillomaviruses are widely distributed in nature and are common in higher vertebrates. The most well-studied is BPV-1, whose genome is a small circular double-stranded DNA that can maintain 20-300 copies in cells. The E1 and E2 proteins encoded by BPV-1, as well as the replication initiation sequence, are necessary to maintain the stable existence of the virus. According to this principle, episomal vectors can be made to stably express foreign genes.

The first virus-based episomal vector was a vector derived from polyomavirus simian virus 40 (SV40). This vector contains the replication initiation sequence carried by SV40 necessary for DNA replication and the large T antigen it encodes. The replication of SV40-derived episomal vectors is not controlled by the host cell, and the copy number can reach thousands, which will lead to cell death.

In the past 10 years, a S/MAR (Scaffold/Matrix Attachment Region, S/MAR)-mediated non-viral attachment carrier has been developed. The matrix attachment region (MAR), also known as the scaffold attachment region (SAR) sequence, is a DNA sequence that remains attached to the nuclear matrix after digestion with restriction enzymes, with a length of about 200-2 000 bp; MAR is enriched in AT base pairs (>70%). MAR elements have three functional features in episomal expression vectors, which can mediate the extrachromosomal attachment of the vector, overcome the silencing of transgene expression and stabilize the vector in mitosis.

Genome editing technology has greatly expanded the ability to modify genes, but the problem of low editing efficiency hinders the application of this technology. The editing efficiency can reach about 30% in easy-to-edit HEK293 cells, but the efficiency is lower than 10% in some difficult-to-transfect cells, which brings trouble for the subsequent selection of two gene copies for deletion. At present, the gene editing process is mainly completed by the transient expression of the edited gene, and the editing efficiency is detected two days after the expression plasmid is transfected. As the cells proliferate without the plasmid proliferating over time, the plasmid is diluted and editing efficiency is reduced. In addition, some cells do not get the plasmid, which will also affect the overall editing efficiency.

Contents of the invention

The purpose of the present invention is to provide a high-efficiency genome editing method and kit for modifying cells, so as to realize high-efficiency editing of cells cultured in vitro.

The genome editing method for modifying cells provided by the present invention uses an attachment carrier encoding a targeted endonuclease, and uses the attachment carrier to express a targeted endonuclease; the attachment carrier can realize non-integrated and stable Gene expression and screening genes are expressed at the same time. Cells that have not obtained the plasmid can be removed through drug screening; when the drug is removed after editing, the cells will gradually lose the attachment carrier, thus obtaining an edited cell line that does not contain foreign genes. The specific steps are:

First, construct an episomal vector encoding a targeting endonuclease and a selection gene (also known as a resistance gene);

Then, the attachment carrier is introduced into the cells, and the cells are cultured for several days under drug selection, so that all cells contain the attachment carrier and stably express the targeting endonuclease.

The principle of improving the editing efficiency of the method of the present invention lies in: (1) the cells containing the episomal carrier are enriched through drug screening; (2) the long-term culture increases the time of gene editing, thereby improving the editing efficiency. the

In the present invention, the attachment carrier can be Epstein-Barr virus (EBV), BK virus (BKV), bovine papilloma virus 1 (BPV-1) or simian virus 40 (SV40) and other viral attachment carriers, and can also be S/MAR (Scaffold/Matrix Attachment Region, S/MAR) mediated non-viral attachment carrier. The common feature of these DNA vectors is that they can replicate and amplify in eukaryotic cells.

In the present invention, the targeted endonuclease may be zinc finger nuclease (ZFN), meganuclease, transcription activator-like effector nuclease (TALEN) or clustered regularly interspaced short palindromic repeat CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats).

In the present invention, the targeted endonuclease cuts DNA at a specific position, and the cell's DNA repair system repairs the DNA break through a non-homologous end-joining repair method or a homologous recombination repair method. Non-homologous end-joining repair often produces base pair additions or deletions, resulting in frameshift mutations in genes. The homologous recombination repair method needs to provide an additional single-stranded or double-stranded donor DNA template that is homologous to both sides of the DNA break site, and the information encoded on the DNA template will be copied to the editing site. This method can precisely change the gene sequence, including the introduction or correction of mutation sites, or site-specific insertion of transgenes. the

In the present invention, the screening gene may be puromycin, neomycin, bleomycin, hygromycin and the like.

In the present invention, the cells may be cultured cells, primary cells, immortal cells, stem cells, induced pluripotent stem cells (iPS) or single-cell embryos. the

In the present invention, the cells may be human cells, mammalian cells, vertebrate cells, invertebrate cells or unicellular eukaryotes.

Suitable cells include: fungi or yeast, such as Pichia, Saccharomyces or Schizosaccharomyce; insect cells, such as SF9 cells from Spodoptera frugiperda or from Drosophila melanogaster (Drosophila melanogaster) S2 cells; animal cells such as mouse, rat, hamster, non-human primate or human cells.

Exemplary cells are mammalian cells. Mammalian cells can be primary cells. In general, any primary cell that is sensitive to double-strand breaks can be used. These cells can be of various cell types, e.g., fibroblasts, myoblasts, T or B cells, macrophages, epithelial cells, etc. Cell lines can be adherent or non-adherent, or can be grown under conditions that stimulate adherent, non-adherent, or organotypic growth using standard techniques known to those skilled in the art. Non-limiting examples of suitable mammalian cell lines include Chinese hamster ovary (CHO) cells, monkey kidney CVI line (COS7) transformed by SV40, human embryonic kidney line 293, baby hamster kidney cells (BHK), mouse Sertoli cells (TM4), monkey kidney cells (CVI-76), African green monkey kidney cells (VERO), human cervical cancer cells (HeLa), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells ( W138), human hepatocytes (Hep G2), mouse mammary tumor cells (MMT), rat hepatoma cells (HTC), HIH/3T3 cells, human U2-OS osteosarcoma cells, human A549 cells, human K562 cells, Human HEK293 cells, human HEK293T cells, human HCT116 cells, human MCF-7 cells and TRI cells.

For the attachment carrier Epstein-Barr virus (i.e. the EBV system), the present invention specifically constructs the following plasmids: EBNA1 Cas9eGFP, EBNA1 Cas9copGFP, EBNA1 Cas9TK, EBNA1 Cas9nTK; wherein:

(1) EBNA1 Cas9eGFP attachment vector, containing hSpCas9 endonuclease, guide RNA expression system (including hU6 promoter, backbone RNA behind and gRNA insertion site between them, namely SapI site), puromycin (Puro ), green fluorescent protein eGFP, elements EBNA1 and EBV oriP for plasmid replication in eukaryotic cells, ampicillin resistance gene (Amp) for amplifying plasmids in E. coli and replication origin pUC origin.

(2) EBNA1 Cas9copGFP attachment vector, containing hSpCas9 endonuclease, guide RNA expression system (including hU6 promoter, the following backbone RNA and the gRNA insertion site between them, that is, the SapI site), puromycin (Puro ), fluorescent protein copGFP (copGFP and puromycin are connected by P2A sequence), elements EBNA1 and EBV oriP for plasmid replication in eukaryotic cells, ampicillin resistance gene (Amp) for amplification of plasmid in E. coli and replication promoter The starting point is pUC origin.

(3) The EBNA1 Cas9TK episomal vector provided by the present invention contains hSpCas9 endonuclease, guide RNA expression system (hU6 promoter gRNA insertion site, that is, SapI site, followed by backbone RNA), puromycin and type 1 simple Fusion protein of herpesvirus thymidine kinase HSV1- tk (purotk), elements EBNA1 and EBV oriP for plasmid replication in eukaryotic cells, ampicillin resistance gene (Amp) for amplification of plasmid in E. coli and replication promoter The starting point is pUC origin.

(4) EBNA1 Cas9nTK attachment vector, containing hSpCas9 endonuclease mutant hSpCas9n (produced but nick-cut), guide RNA expression system (hU6 promoter gRNA insertion site, that is, SapI site, followed by backbone RNA), purine Fusion protein of mymycin and herpes simplex virus type 1 thymidine kinase HSV1- tk (purotk), elements EBNA1 and EBV oriP for plasmid replication in eukaryotic cells, ampicillin resistance gene for amplification of plasmids in Escherichia coli (Amp) and the origin of replication pUC origin.

The specific steps of the EBNA1 Cas9eGFP attachment vector construction method are:

(1) Digest the pREP9 plasmid with XbaI and KpnI, and retain the part expressing the EBNA1 gene.

(2) The SV40pA sequence was artificially synthesized and cloned into the KpnI and XbaI sites of the pREP9 plasmid to obtain the plasmid pREP9-pA. The specific sequence is:

GGTACC TTTCAGA GCGATCGC AAGTAA CTTAAG CTTAAGAACCGCTCGAGGCCGGCAAGGCCGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGAGGTCGAC TCTAGA （SEQ.ID.NO.1）。

The underlined parts are restriction enzyme cutting sites KpnI, AsiSI, AflII and XbaI respectively.

(3) The plentiCRISPR plasmid (from Feng Zhang's laboratory in the United States) was digested with BsmBI and replaced with double SapI restriction sites. The double SapI restriction site was formed by annealing oligo CACCGAGAAGAGCGATGCTCTTCG (SEQ.ID.NO.2) and AAACCGAAGAGCATCGCTCTTCTC (SEQ.ID.NO.3), which could just connect to the BsmBI restriction site to obtain the plasmid plentiCRISPR-SapI.

(4) Use primers TATGGTACCTTTCAGACCCACCTCCCAAC (SEQ.ID.NO.4) and CCTTCGGCGATCGCGGCACCGGGCTTGCGGGTCA (SEQ.ID.NO.5) to amplify the Cas9 part from plentiCRISPR-SapI, digest with KpnI and AsiSI, and connect to pREP9-pA.

(5) Use primers GGTGCCGCGATCGCCGAAGGATCCGCGGCCGCCA (SEQ.ID.NO.6) and GTTCTTAAGTTACTTGTACAGCTCGTCCA (SEQ.ID.NO.7) to amplify eGFP from the peGFP-N1 plasmid, digest with AsiSI and AflII, and connect to the above (4) The plasmid obtained in the obtained EBNA1 Cas9eGFP episome vector. the

The specific steps of the EBNA1 Cas9copGFP attachment vector construction method are:

Use primers GCCGCGATCGCCGAAGGATCCGCGGCCGCTG (SEQ.ID.NO.8) and GTTCTTAAGTCGACTTAGATTCAGCTCTA (SEQ.ID.NO.9) to amplify the P2AcopGFP part from the pCDH_EF1_MCS_T2A_copGFP (System Biosciences) plasmid, digest with AsiSI and AflII, and connect to the Cas9eGFP plasmid of EBNA1 , AflII restriction site, to obtain the EBNA1 Cas9copGFP attachment vector.

The specific steps of the EBNA1 Cas9TK attachment vector construction method are:

Use primers ATCCGGACCGCCACATCGAGCGGGTCACCG (SEQ.ID.NO.10) and GTTCTTAAGGCTGATCAGCGAGCTCTAGA (SEQ.ID.NO.11) to amplify the part of puroTK from the pT2/RMCE-OSKM-puDTk (PB ITR deleted) plasmid, digest with RsrII and AflII, Connect to the RsrII and AflII restriction sites of the EBNA1 Cas9eGFP plasmid to obtain the EBNA1 Cas9TK attachment vector.

The specific steps of the EBNA1 Cas9nTK attachment vector construction method are:

The 29th base of hSpCas9 encoded by EBNA1 Cas9TK was changed from A to C by the point mutation method to obtain the EBNA1 Cas9nTK attachment vector.

Regarding other attachment vectors: BKV, BPV-1, SV40, S/MAR, they can be constructed by similar methods as above according to actual needs.

The present invention further provides a method for editing cell chromosomes using the EBNA1 Cas9eGFP, EBNA1 Cas9copGFP, EBNA1 Cas9TK and EBNA1 Cas9nTK attachment carrier systems. The use methods of these four attachment carrier systems are the same, so the EBNA1 Cas9eGFP attachment carrier is specifically used for description. Guide RNA was designed according to the editing site, and a pair of oligos were synthesized. The oligos were annealed to form double-stranded DNA, and the ends matched with the cohesive ends generated by SapI digestion of the EBNA1 Cas9TeGFP attachment vector. The double-stranded DNA fragment was ligated to the EBNA1 Cas9eGFP attachment vector with T4 DNA ligase to obtain EBNA1 Cas9eGFP-gRNA. Introduce EBNA1 Cas9eGFP-gRNA into the cells to be edited. The introduction method can be conventional cell transfection reagents, such as Lipofectamin2000, or electrotransfection. Two days after transfection, the cells were screened with an appropriate concentration of resistance gene (also known as selection gene), and the cells that were not transfected were killed. Different cells require different concentrations of resistance genes, so it is necessary to achieve concentration titration and try to use low doses of drugs for screening. The timing of editing will vary depending on the editing site and cell type. Normally it takes 8-30 days. The method for detecting editing efficiency can use the T7 enzyme or Cel-1 enzyme digestion method often used in this field. After the editing is completed, the resistance gene is removed, and the cells are isolated and cultured for several days by flow cytometry, and the DNA is extracted, and the PCR editing site is connected to the T plasmid vector for sequencing. When cultured without resistance gene selection, episomal vectors are gradually lost from the cells. When using EBNA1 Cas9TK and EBNA1 Cas9nTK episomal vectors, remove the resistance gene and culture for 3 days after editing, some cells will lose the episomal plasmid during proliferation, and then add ganciclovir for 5 days to kill cells containing the edited plasmid. cells, so that cell lines free of editing plasmids can be quickly obtained.

Episomal vectors need to encode resistance genes to function in eukaryotic cells. During the transfection process, some cells did not get the plasmid, and these cells need to be killed by drug screening, and only the cells that got the plasmid were allowed to survive. On the other hand, some cells will lose the attachment plasmid during division, so it is also necessary to rely on drugs to kill these cells. The resistance gene can be puromycin, neomycin, bleomycin, hygromycin and the like.

Where homologous recombination is required to provide donor DNA with homology to the restriction site, the ratio of episomal plasmid targeting endonuclease molecules to donor vector can and will vary. Typically, the ratio of episomal plasmid to donor plasmid can range from about 1:10 to about 10:1. In various embodiments, the ratio of episomal plasmid to donor plasmid can be about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1. the

The frequency of targeted editing of chromosomal sequences can and will vary according to a number of factors. In some embodiments, the frequency of editing may be greater than about 0.1%, 0.3%, 1%, 3%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% % or 100%. Single cell clones comprising edited chromosomal sequences can be isolated using techniques well known in the art. Those of skill in the art are familiar with methods for generating cells homozygous for an edited chromosomal sequence. In other words, cells can be heterozygous or homozygous for the edited chromosomal sequence.

The present invention also provides a kit for constructing an episomal vector gene editor, which contains:

(1) Attachment vectors encoding targeting endonucleases and screening genes;

(2) PCR primers for detection of editing sites;

(3) T4 ligase and ligation buffer.

In the kit of the present invention, the attachment body carrier encoding the targeting endonuclease and the screening gene can be one of the above-mentioned EBV, BKV, BPV-1, SV40, S/MAR, the DNA carrier of this type A common feature is the ability to replicate and amplify in eukaryotic cells.

In the kit of the present invention, the targeted endonuclease may be the above-mentioned zinc finger nuclease (ZFN), meganuclease, transcription activator-like effector nuclease (TALEN) or regularly clustered interspaced short palindromic repeats One of CRISPR/Cas9.

In the kit of the present invention, the attachment carrier Epstein-Barr virus (i.e. EBV system) can specifically be several plasmids constructed above: EBNA1 Cas9eGFP, EBNA1 Cas9TK, EBNA1 Cas9TK, EBNA1 Cas9nTK.

The invention is applicable to genome editing of various eukaryotic cells.

Description of drawings

Fig. 1 is four attachment carrier structures of the present invention. Among them, Figure 1A shows the structure of the EBNA1 Cas9eGFP attachment vector, which is an attachment DNA expressing a guide RNA (gRNA), a Cas9 protein, a puromycin resistance gene (Puro) and a green fluorescent protein (eGFP) (EBV) system. Figure 1B shows the structure of the EBNA1 Cas9copGFP episomal vector, which is an episomal DNA (EBV) expressing a guide RNA (gRNA), a Cas9 protein, a puromycin resistance gene (Puro), and a fluorescent protein (copGFP) system. Figure 1C shows the structure of the EBNA1 Cas9TK attachment vector, which is a fusion expressing a guide RNA (gRNA), a Cas9 protein, a puromycin and resistance gene, and herpes simplex virus type 1 thymidine kinase HSV1- tk Protein (purotk) episome vector (EBV) system. Figure 1D shows the structure of the EBNA1 Cas9nTK attachment vector. Compared with the EBNA1 Cas9TK attachment vector, it only has a D10A mutation in the Cas9 coding region, and the rest are the same. oriP is the replication initiation site sequence of the plasmid in eukaryotic cells, and the protein encoded by EBNA1 interacts with the oriP sequence to initiate the replication of the plasmid. Ori is an origin of replication sequence functional in prokaryotes; Amp is an ampicillin resistance gene.

Figure 2 shows the results of editing the AAVS1 site with the EBNA1 Cas9eGFP attachment vector in HEK293 cells. Figure 2A is the sequence of the editing site detected by PCR, the underlined part is the gRNA recognition sequence, the red mark is the PAM sequence, and the green part is the SacI restriction site. Figure 2B is a SacI digested gel electrophoresis image of the PCR product, which is used to detect the editing efficiency. If the site is edited, the SacI site will be destroyed, resulting in the inability of SacI to cut. Figure 2C is a quantitative graph of editing efficiency, the vertical axis is the percentage of edited DNA in total DNA, and the horizontal axis is the number of days of editing.

Figure 3 shows the results of editing the EMX1 site with the EBNA1 Cas9copGFP attachment vector in HEK293 cells. Figure 3A is the sequence of the editing site detected by PCR. The underlined part is the gRNA recognition sequence, the red mark is the PAM sequence, and the green and red parts are the AgeI restriction site. Figure 3B is an AgeI digested gel electrophoresis image of the PCR product, which is used to detect the editing efficiency. If the site is edited, the AgeI site is destroyed, resulting in failure of AgeI to cut. Figure 3C is a quantitative graph of editing efficiency, the vertical axis is the percentage of edited DNA in total DNA, and the horizontal axis is the number of days of editing.

Figure 4 shows the results of editing the GRIN2B site with the EBNA1 Cas9TK attachment vector in HEK293 cells. Figure 4A is the sequence of the editing site detected by PCR, the underlined part is the gRNA recognition sequence, the red mark is the PAM sequence, and the green part is the MSCI restriction site. Figure 4B is an MSCI digested gel electrophoresis image of the PCR product, which is used to detect the editing efficiency. If the site is edited, the MSCI site will be destroyed, resulting in the inability of MSCI to cut. Figure 4C is a quantitative graph of editing efficiency, the vertical axis is the percentage of edited DNA in the total DNA, and the horizontal axis is the number of days of editing.

Figure 5 shows the results of editing the DYRK1A site with the EBNA1 Cas9eGFP attachment vector in HEK293 cells. Figure 5A is the sequence of the editing site detected by PCR, the underlined part is the gRNA recognition sequence, the red mark is the PAM sequence, and the green and red parts are the BstXI restriction site. Figure 5B is the DYRK1A digested gel electrophoresis image of the PCR product, which is used to detect the editing efficiency. If the site is edited, the DYRK1A site is destroyed, resulting in the inability of DYRK1A to cut. Figure 5C is a quantitative graph of editing efficiency, the vertical axis is the percentage of edited DNA to the total DNA, and the horizontal axis is the number of days of editing.

Figure 6 shows a system for simultaneous expression of two guide RNAs (gRNAs) using the EBNA1 Cas9eGFP episomal vector for simultaneous editing of two genomic loci. oriP is the replication initiation site sequence of the plasmid in eukaryotic cells, and the protein encoded by EBNA1 interacts with the oriP sequence to initiate the replication of the plasmid. Ori is an origin of replication sequence functional in prokaryotes; Amp is an ampicillin resistance gene.

Figure 7 shows the results of EBNA1 Cas9eGFP attachment vector editing DYRK1A and EMX1 sites in HEK293 cells. Figure 7A is the gel electrophoresis image of DYRK1A digestion of PCR products, which is used to detect the editing efficiency. If the site is edited, the DYRK1A site is destroyed, resulting in the inability of DYRK1A to cut. Figure 7B is a quantitative graph of editing efficiency, the vertical axis is the percentage of edited DNA in the total DNA, and the horizontal axis is the number of days of editing. Figure 7C is the EMX1 digested gel electrophoresis image of the PCR product, which is used to detect the editing efficiency. If the site is edited, the EMX1 site is destroyed, making it impossible for EMX1 to cut. Figure 7D is a quantitative graph of editing efficiency, the vertical axis is the percentage of edited DNA in the total DNA, and the horizontal axis is the number of days of editing.

Figure 8 shows the episomal system using the EBNA1 Cas9nTK episomal vector to simultaneously express two guide RNAs (gRNAs), which are in close proximity and generate DNA double-strand breaks through double nicks. oriP is the replication initiation site sequence of the plasmid in eukaryotic cells, and the protein encoded by EBNA1 interacts with the oriP sequence to initiate the replication of the plasmid. Ori is an origin of replication sequence functional in prokaryotes; Amp is an ampicillin resistance gene.

Figure 9 shows the results of editing the EMX1 site with the plasmid in Figure 8 in HEK293 cells. The EMX1 site is the same as that described in Figure 3. The underlined part in Figure 9A indicates the two gRNA sequences, and the green part is the AgeI restriction site. Figure 9B is the EMX1 digested gel electrophoresis image of the PCR product, which is used to detect the editing efficiency. If the site is edited, the EMX1 site is destroyed, making it impossible for EMX1 to cut. Figure 9C is a quantitative graph of editing efficiency, the vertical axis is the percentage of edited DNA in the total DNA, and the horizontal axis is the number of days of editing.

Figure 10 is the result of deleting a 281 base pair of the EMX1 site with the EBNA1 Cas9eGFP plasmid in Figure 6 in HEK293 cells. The underlined portion of Figure 10A indicates the two gRNA sequences. Fig. 10B is a PCR gel electrophoresis image of detection deletion results. Deletion fragments were detectable on the third day and increased over time.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. This embodiment is only used to illustrate the present invention

It is not construed as a limitation of the present invention.

Example 1: Editing of the AAVS1 locus using the EBNA1 Cas9eGFP episome vector

The following example details the use of the EBNA1 Cas9eGFP attachment vector to edit the AAVS1 locus, the sequence of the target site is 5'- AAGAAGACTAGCTGAGCTCTCGG -3' (SEQ.ID.NO.12), and the final CGG sequence is necessary for CRISPR/Cas9 editing The PAM sequence. 200,000 human HEK293 cells were transfected with 1 μg of the EBNA1 Cas9eGFP plasmid described in Figure 1A. After one day of incubation, selection was performed with 2 mg/ml puromycin. On the 1st, 3rd, 6th, 9th and 12th day, DNA was extracted to identify the efficiency of digestion. The site of CRISPR/Cas9 editing has a SacI cleavage site (GAGCTC), which has been marked in green (Fig. 2A). After editing occurs, this site is destroyed, and the PCR product cannot be cut by SacI, so the efficiency can be identified by enzyme digestion. It can be seen from Figure 2B that the editing did not occur on the first day of transfection, and the target site could be completely cut by SacI. As time went by, more and more target sites were edited. By the ninth day, almost 100% The target site is edited.

Example 2: Editing of the EMX1 locus using the EBNA1 Cas9copGFP episome vector

The following examples detail editing of the EMX1 locus using the EBNA1 Cas9copGFP episomal vector. The guide RNA for EMX1 was expressed with the EBNA1 Cas9copGFP episomal vector as shown in Figure 1B. The sequence of the target site is 5'- AGGGCTCCCATCACATCAACCGG -3' (SEQ.ID.NO.13), and the last CGG sequence is the PAM sequence necessary for CRISPR/Cas9 editing. 200,000 human HEK293 cells were transfected with 1 μg of the plasmid described in Figure 1A. After one day of incubation, selection was performed with 2 mg/ml puromycin. On the 1st, 3rd, 6th, 9th, 12th, and 18th days, DNA was extracted to identify the enzyme digestion efficiency. The site of CRISPR/Cas9 editing has an AgeI restriction site (ACCGGT), which has been marked in green (Fig. 3A). After editing occurs, this site is destroyed, and the PCR product cannot be cut by AgeI, so the efficiency can be identified by enzyme digestion. It can be seen from Figure 3B that the editing did not occur on the first day of transfection, and the target site could be completely cut by AgeI. As time went by, more and more target sites were edited. By the 18th day, almost 100% The target site is edited.

Example 3: Editing of the GRIN2B locus using the EBNA1 Cas9TK episomal vector

The following examples detail editing of the GRIN2B gene using the EBNA1 Cas9TK episomal vector. The guide RNA for GRIN2B was expressed with the EBNA1 Cas9TK episome vector as shown in Figure 1C. The sequence of the target site is 5'-TTCCGACGAGGTGGCCATCAAGG -3' (SEQ.ID.NO.14), and the last AGG sequence is the PAM sequence necessary for CRISPR/Cas9 editing. 200,000 human HEK293 cells were transfected with 1 μg of the plasmid described in Figure 1A. After one day of incubation, selection was performed with 2 mg/ml puromycin. On the 1st, 3rd, 6th, 9th, 12th, 18th and 22nd days, DNA was extracted to identify the enzyme digestion efficiency. The site of CRISPR/Cas9 editing has an MSCI restriction site (TGGCCA), which has been marked in green (Fig. 4A). After editing, this site is destroyed, and the PCR product cannot be cut by MSCI, so the efficiency can be identified by enzyme digestion. It can be seen from Figure 4B that editing did not occur on the first day of transfection, and the target site could be completely cut by MSCI. As time went on, more and more target sites were edited. By the 22nd day, 65% of the target sites The site is edited.

Example 4: Editing of the DYRK1A locus using the EBNA1 Cas9eGFP attachment vector

The following examples detail editing of the DYRK1A locus. The guide RNA for DYRK1A was expressed with the EBNA1 Cas9eGFP attachment vector as shown in Figure 1A. The sequence of the target site is 5'- CCATCTGAAGGCCAGCAGCATGG -3' (SEQ.ID.NO.15), and the last TGG sequence is the PAM sequence necessary for CRISPR/Cas9 editing. 200,000 human HEK293 cells were transfected with 1 μg of the plasmid described in Figure 1A. After one day of incubation, selection was performed with 2 mg/ml puromycin. On the 1st, 3rd, 6th, 9th, 12th, 18th and 22nd days, DNA was extracted to identify the enzyme digestion efficiency. The site of CRISPR/Cas9 editing has a BstXI restriction site (CCAGCAGACTGG), which has been marked in green (Fig. 5A). After editing, this site is destroyed, and the PCR product cannot be cut by BstXI, so the efficiency can be identified by enzyme digestion. It can be seen from Figure 5B that the editing did not occur on the first day of transfection, and the target site could be completely cut by BstXI. As time went by, more and more target sites were edited. By the 12th day, about 100% The target site is edited.

Example 5: Simultaneous editing of the DYRK1A and EMX1 loci using the EBNA1 Cas9eGFP attachment vector

The following examples detail simultaneous editing of DYRK1A and EMX1 loci in one cell. Use the EBNA1 Cas9eGFP attachment vector shown in Figure 6 to simultaneously express the guide RNAs at the DYRK1A and EMX1 sites. 200,000 human HEK293 cells were transfected with 1 μg of the plasmid described in Figure 6. After one day of incubation, selection was performed with 2 mg/ml puromycin. On the 1st, 3rd, 6th, 9th, 12th, 15th, 18th and 22nd day, DNA was extracted to identify the enzyme digestion efficiency. It can be seen from Figure 7A that the editing efficiency of the DYRK1A site was close to 100% on the 18th day after transfection, and 90% of the EMX1 target site editing was completed on the 22nd day after transfection.

Example 6: Editing of the EMX1 locus using the EBNA1 Cas9nTK episomal vector

The following examples detail editing of the EMX1 locus using double nicking. As shown in Figure 8, the EBNA1 Cas9nTK episome vector expresses two guide RNAs for EMX1. The Cas9 endonuclease expressed by this vector contains a D10A mutation, which can only cut DNA single strands at a specific point under the guidance of the guide RNA, and two single-strand breaks with a suitable distance can form a DNA double-strand break. This approach requires simultaneous cleavage of both target sites to generate mutations, thus enhancing the specificity of cleavage. The sequences of the target sites were 5'-TGCGCCACCGGTTGATGTGATGG-3' (SEQ.ID.NO.16) and 5'-CACGAAGCAGGCCAATGGGGAGG-3' (SEQ.ID.NO.17) (see Figure 9A). 200,000 human HEK293 cells were transfected with 1 μg of the plasmid described in Figure 8. After one day of incubation, selection was performed with 2 mg/ml puromycin. On the 1st, 3rd, 6th, 9th, 12th, 15th, 18th and 22nd day, DNA was extracted to identify the enzyme digestion efficiency. The site of CRISPR/Cas9 editing has an AgeI restriction site (ACCGGT), which has been marked in green (Fig. 9A). After editing occurs, this site is destroyed, and the PCR product cannot be cut by AgeI, so the efficiency can be identified by enzyme digestion. It can be seen from Figure 9B that no editing occurred on the first day of transfection, and 80% of the target site editing was completed on the 22nd day.

Embodiment 7: Utilize EBNA1 Cas9eGFP attachment carrier to delete 281 base fragments at EMX1 locus

The following examples detail the use of the EBNA1 Cas9eGFP attachment vector to knock out a 281-base fragment at the EMX1 locus. As shown in Figure 6, the EBNA1 Cas9eGFP attachment vector expresses two guide RNAs of EMX1, and the sequence of the target site is 5'-AGGCCCCAGTGGCTGCTCTGGGG-3'(SEQ.ID.NO.18) and 5'-GGCAGAGTGCTGCTTGCTGCTGG-3'( SEQ.ID.NO.19) (see Figure 10A). 200,000 human HEK293 cells were transfected with 1 μg of the plasmid described in Figure 8. After one day of incubation, selection was performed with 2 mg/ml puromycin. On the 1st, 3rd, 6th, 9th, 12th, 15th, 18th, 21st, 24th, 27th and 30th days, DNA was extracted and PCR was used to preliminarily identify the enzyme cutting efficiency. From Figure 10B, it can be seen that the editing did not occur on the first day of transfection, the deletion of DNA fragments could be detected on the third day, and the editing was basically completed after the 12th day, but there were still some unedited DNA bands.

Primers for detecting the AAVS1 site in the present invention:

TGCTTTCTTTGCCTGGACAC (SEQ.ID.NO.20)

CTGTCACCAATCCTGTCCCT (SEQ.ID.NO.21);

Primers for detecting the EMX1 site in the present invention:

CCATCCCCTTCTGTGAATGT (SEQ.ID.NO.22)

GGAGATTGGAGACACGGAGA (SEQ. ID. NO. 23);

Primers for detecting the DYRK1A site in the present invention:

GGAGCTGGTCTGTTGGAGAA (SEQ.ID.NO.24)

TCCCAATCCATAATCCCACGTT (SEQ.ID.NO.25);

Primers for detecting the GRIN2B site in the present invention:

CAGGAGGGCCAGGAGATTTG (SEQ.ID.NO.26)

TGAAATCGAGGATCTGGGCG (SEQ. ID. NO. 27).

<110> Fudan University

<120> Genome editing methods and kits using vectors encoding targeted endonuclease attachments

<130> 001

<160> 27

<170> PatentIn version 3.3

the

<210> 1

<211> 472

<212> DNA

<213> 

the

<400> 1

ggtacctttc agagcgatcg caagtaactt aagcttaaga accgctcgag gccggcaagg 60

the

ccggatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag 120

the

tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 180

the

agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 240

the

gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat ggctgattat 300

the

gatccggctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc 360

the

cggagacggt cacagcttgt ctgtaagcgg atgccggggag cagacaagcc cgtcagggcg 420

the

cgtcagcggg tgttggcggg tgtcggggcg cagccatgag gtcgactcta ga 472

the

the

<210> 2

<211> 24

<212> DNA

<213> 

the

<400> 2

caccgagaag agcgatgctc ttcg 24

the

the

<210> 3

<211> 24

<212> DNA

<213> 

the

<400> 3

aaaccgaaga gcatcgctct tctc 24

the

the

<210> 4

<211> 29

<212> DNA

<213> 

the

<400> 4

tatggtacct ttcagaccca cctcccaac 29

the

the

<210> 5

<211> 34

<212> DNA

<213> 

the

<400> 5

ccttcggcga tcgcggcacc gggcttgcgg gtca 34

the

the

<210> 6

<211> 34

<212> DNA

<213> 

the

<400> 6

ggtgccgcga tcgccgaagg atccgcggcc gcca 34

the

the

<210> 7

<211> 29

<212> DNA

<213> 

the

<400> 7

gttcttaagt tacttgtaca gctcgtcca 29

the

the

<210> 8

<211> 31

<212> DNA

<213> 

the

<400> 8

gccgcgatcg ccgaaggatc cgcggccgct g 31

the

the

<210> 9

<211> 29

<212> DNA

<213> 

the

<400> 9

gttcttaagt cgacttagat tcagctcta 29

the

the

<210> 10

<211> 30

<212> DNA

<213> 

the

<400> 10

atccggaccg ccacatcgag cgggtcaccg 30

the

the

<210> 11

<211> 29

<212> DNA

<213> 

the

<400> 11

gttcttaagg ctgatcagcg agctctaga 29

the

the

<210> 12

<211> 23

<212> DNA

<213> 

the

<400> 12

aagaagacta gctgagctct cgg 23

the

the

<210> 13

<211> 23

<212> DNA

<213> 

the

<400> 13

agggctccca tcacatcaac cgg 23

the

the

<210> 14

<211> 23

<212> DNA

<213> 

the

<400> 14

ttccgacgag gtggccatca agg 23

the

the

<210> 15

<211> 23

<212> DNA

<213> 

the

<400> 15

ccatctgaag gccagcagca tgg 23

the

the

<210> 16

<211> 23

<212> DNA

<213> 

the

<400> 16

tgcgccaccg gttgatgtga tgg 23

the

the

<210> 17

<211> 23

<212> DNA

<213> 

the

<400> 17

cacgaagcag gccaatgggg agg 23

the

the

<210> 18

<211> 23

<212> DNA

<213> 

the

<400> 18

aggccccagt ggctgctctg ggg 23

the

the

<210> 19

<211> 23

<212> DNA

<213> 

the

<400> 19

ggcagagtgc tgcttgctgc tgg 23

the

the

<210> 20

<211> 20

<212> DNA

<213> 

the

<400> 20

tgctttcttt gcctggacac 20

the

the

<210> 21

<211> 20

<212> DNA

<213> 

the

<400> 21

ctgtcaccaa tcctgtccct 20

the

the

<210> 22

<211> 20

<212> DNA

<213> 

the

<400> 22

ccatcccctt ctgtgaatgt 20

the

the

<210> 23

<211> 20

<212> DNA

<213> 

the

<400> 23

ggagattgga gacacggaga 20

the

the

<210> 24

<211> 20

<212> DNA

<213> 

the

<400> 24

ggagctggtc tgttggagaa 20

the

the

<210> 25

<211> 22

<212> DNA

<213> 

the

<400> 25

tcccaatcca taatcccacg tt 22

the

the

<210> 26

<211> 20

<212> DNA

<213> 

the

<400> 26

caggagggcc aggagatttg 20

the

the

<210> 27

<211> 20

<212> DNA

<213> 

the

<400> 27

tgaaatcgag gatctgggcg 20

the

the

Claims (11)

1., for a genome edit methods for modified cells, it is characterized in that concrete steps are:

First, an attachment vector encoded target endonuclease and screening-gene is built;

Then, by described attachment vector introduction in cell, the some skies of culturing cell under the screening of medicine, make all to contain attachment carrier in all cells and stably express target endonuclease.

2., according to the genome edit methods described in claim 1, it is characterized in that described attachment carrier is viral attached type carrier: EBV, BKV, BPV-1 or SV40, or non-viral attached type carrier: S/MAR; The common trait of this kind of DNA vector can copy amplification in eukaryotic cell.

3. the genome edit methods according to claim 1 or 2, is characterized in that described target endonuclease is that the short palindrome in Zinc finger nuclease, meganuclease, transcriptional activator sample effector nuclease or rule cluster interval repeats CRISPR/Cas9.

4. genome edit methods according to claim 3, is characterized in that described target endonuclease cuts off DNA at specific position, and the DNA repair system of cell is by non-homologous end joining repairing method or the fracture of homologous recombination repair method DNA plerosis.

5., according to the genome edit methods described in claim 1, it is characterized in that described cell is cultured cells, primary cell, immortality cell, stem cell, the pluripotent stem cell of induction or slender blastula.

6., according to the genome edit methods described in claim 1, it is characterized in that described cell is people's cell, mammalian cell, vertebrate cells, invertebral zooblast or unicellular eukaryote.

7. according to the genome edit methods described in claim 6, it is characterized in that described mammalian cell comprises Chinese hamster ovary cell, the monkey kidney CVI system transformed by SV40, human embryo kidney (HEK) system 293, baby hamster kidney cell, mouse Sertoli cell, monkey-kidney cells, African green monkey kidney cell, human cervical carcinoma cell, Madin-Darby canine kidney(cell line), buffalo rat hepatocytes, human pneumonocyte, human liver cell, mammary gland of mouse oncocyte, rats'liver oncocyte, HIH/3T3 cell, people U2-OS osteosarcoma cell, people A549 cell, people K562 cell, people HEK293 cell, people HEK293T cell, people HCT116 cell, people MCF-7 cell and TRI cell.

8., according to the genome edit methods described in claim 3, it is characterized in that described attachment carrier EBV system, the one for following several plasmid: EBNA1 Cas9eGFP, EBNA1 Cas9copGFP, EBNA1 Cas9TK, EBNA1 Cas9nTK; Wherein:

(1) EBNA1 Cas9eGFP attachment carrier, contain: hSpCas9 restriction endonuclease, guide rna expression system, comprise: hU6 promotor, skeleton RNA below and the gRNA insertion point between them and SapI site, tetracycline, the element EBNA1 that green fluorescent protein eGFP, plasmid copy in eukaryotic cell and EBV oriP, the ammonia benzyl resistant gene (Amp) of the plasmid that increases in intestinal bacteria and replication origin pUC origin;

(2) EBNA1 Cas9copGFP attachment carrier, contain: hSpCas9 restriction endonuclease, guide rna expression system, comprise: hU6 promotor, skeleton RNA below and the gRNA insertion point between them and SapI site, tetracycline, fluorescin copGFP, the element EBNA1 that plasmid copies in eukaryotic cell and EBV oriP, the ammonia benzyl resistant gene (Amp) of the plasmid that increases in intestinal bacteria and replication origin pUC origin;

(3) EBNA1 Cas9TK attachment carrier, contain: hSpCas9 restriction endonuclease, guide rna expression system: hU6 promotor gRNA insertion point and SapI site, skeleton RNA below, tetracycline and herpes simplex types 1 virus thymidine kinase HSV1- tkthe element EBNA1 that copies in eukaryotic cell of fusion rotein (purotk), plasmid and EBV oriP, the ammonia benzyl resistant gene (Amp) of the plasmid that increases in intestinal bacteria and replication origin pUC origin;

(4) EBNA1 Cas9nTK attachment carrier, contain: the mutant hSpCas9n of hSpCas9 restriction endonuclease, guide rna expression system: hU6 promotor gRNA insertion point and SapI site, skeleton RNA below, tetracycline and herpes simplex types 1 virus thymidine kinase HSV1- tkfusion rotein (purotk), the element EBNA1 that plasmid copies in eukaryotic cell and EBV oriP, the ammonia benzyl resistant gene (Amp) of the plasmid that increases in intestinal bacteria and replication origin pUC origin.

9. genome edit methods according to claim 8, it is characterized in that utilizing EBNA1 Cas9eGFP, EBNA1 Cas9copGFP, EBNA1 Cas9TK and EBNA1 Cas9nTK attachment carrier system editor cell chromosome, wherein, the concrete steps of EBNA1 Cas9eGFP attachment carrier system editor cell chromosome are utilized to be:

According to editing sites Design wizard RNA, synthesize a pair oligo, double-stranded DNA is formed after oligo annealing, and double chain DNA fragment is connected on EBNA1 Cas9eGFP attachment carrier by the sticky end coupling T4 DNA ligase that end and SapI enzyme cut the generation of EBNA1 Cas9TeGFP attachment carrier, obtains EBNA1 Cas9eGFP-gRNA;

Imported to by EBNA1 Cas9eGFP-gRNA in the cell that will edit, transfection is two days later with the resistant gene screening cell of appropriate concentration, and not having the cell of transfection to kill, the time of editor is 8-30 days;

Removed by resistant gene after having edited, cell flow cytometer separates the some skies of Colony Culture, and extraction DNA, PCR editing sites is connected on T plasmid vector and checks order, and obtains not containing editor's clone of foreign gene;

Wherein, described resistant gene is tetracycline, Liu Suanyan NEOMYCIN SULPHATE, bleomycin, or Totomycin;

Utilize EBNA1 Cas9copGFP, EBNA1 Cas9TK and EBNA1 Cas9nTK attachment carrier system editor cell chromosome step, identical with utilizing the step of EBNA1 Cas9eGFP attachment carrier system editor cell chromosome.

10., for editing a test kit for cellular genome, it is characterized in that described test kit comprises:

(1) the attachment carrier of coding target endonuclease and screening-gene;

(2) for detecting the primer of the PCR of editing sites;

(3) T4 ligase enzyme be connected damping fluid;

Wherein, the attachment carrier of described coding target endonuclease and screening-gene is the one in EBV, BKV, BPV-1, SV40, S/MAR, and the common trait of this kind of DNA vector can copy amplification in eukaryotic cell;

Described target endonuclease is the one that the short palindrome in above-mentioned Zinc finger nuclease, meganuclease, transcriptional activator sample effector nuclease or rule cluster interval repeats in CRISPR/Cas9.

11. test kits for editing cellular genome according to claim 10, is characterized in that described attachment carrier EBV system is the one of following plasmid: EBNA1 Cas9eGFP, EBNA1 Cas9TK, EBNA1 Cas9TK, EBNA1 Cas9nT; Wherein:

(1) EBNA1 Cas9eGFP attachment carrier, contain: hSpCas9 restriction endonuclease, guide rna expression system, comprise: hU6 promotor, skeleton RNA below and the gRNA insertion point between them and SapI site, tetracycline, the element EBNA1 that green fluorescent protein eGFP, plasmid copy in eukaryotic cell and EBV oriP, the ammonia benzyl resistant gene (Amp) of the plasmid that increases in intestinal bacteria and replication origin pUC origin;

(2) EBNA1 Cas9copGFP attachment carrier, contain: hSpCas9 restriction endonuclease, guide rna expression system, comprise: hU6 promotor, skeleton RNA below and the gRNA insertion point between them and SapI site, tetracycline, fluorescin copGFP, the element EBNA1 that plasmid copies in eukaryotic cell and EBV oriP, the ammonia benzyl resistant gene (Amp) of the plasmid that increases in intestinal bacteria and replication origin pUC origin;

(3) EBNA1 Cas9TK attachment carrier, contain: hSpCas9 restriction endonuclease, guide rna expression system: hU6 promotor gRNA insertion point and SapI site, skeleton RNA below, tetracycline and herpes simplex types 1 virus thymidine kinase HSV1- tkthe element EBNA1 that copies in eukaryotic cell of fusion rotein (purotk), plasmid and EBV oriP, the ammonia benzyl resistant gene (Amp) of the plasmid that increases in intestinal bacteria and replication origin pUC origin;

(4) EBNA1 Cas9nTK attachment carrier, contain: the mutant hSpCas9n of hSpCas9 restriction endonuclease, guide rna expression system: hU6 promotor gRNA insertion point and SapI site, skeleton RNA below, tetracycline and herpes simplex types 1 virus thymidine kinase HSV1- tkfusion rotein (purotk), the element EBNA1 that plasmid copies in eukaryotic cell and EBV oriP, the ammonia benzyl resistant gene (Amp) of the plasmid that increases in intestinal bacteria and replication origin pUC origin.