WO2020015279A1 - 一种在干细胞中进行基因定向敲入的方法 - Google Patents

一种在干细胞中进行基因定向敲入的方法 Download PDF

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WO2020015279A1
WO2020015279A1 PCT/CN2018/119852 CN2018119852W WO2020015279A1 WO 2020015279 A1 WO2020015279 A1 WO 2020015279A1 CN 2018119852 W CN2018119852 W CN 2018119852W WO 2020015279 A1 WO2020015279 A1 WO 2020015279A1
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gene
knock
homology arm
cell
seq
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PCT/CN2018/119852
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English (en)
French (fr)
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李程
丁秋蓉
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杭州观梓健康科技有限公司
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Priority claimed from CN201810786497.2A external-priority patent/CN109055373B/zh
Priority claimed from CN201810786496.8A external-priority patent/CN108949691B/zh
Priority claimed from CN201810784680.9A external-priority patent/CN109082442B/zh
Priority claimed from CN201810785793.0A external-priority patent/CN109097333B/zh
Priority claimed from CN201810785795.XA external-priority patent/CN109082443A/zh
Priority claimed from CN201810784678.1A external-priority patent/CN108949690B/zh
Application filed by 杭州观梓健康科技有限公司 filed Critical 杭州观梓健康科技有限公司
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present disclosure relates to the field of biotechnology, and in particular, to a method for targeted gene knock-in in stem cells.
  • DSBs precise double-strand breaks
  • DSBs can activate non-homologous end-joining (NHEJ) and homology-directed repair (HDR) repair mechanisms to repair DNA.
  • NHEJ non-homologous end-joining
  • HDR homology-directed repair
  • the NHEJ repair gene predominates, and random insertions or deletions of bases can be introduced to achieve gene knockout.
  • HDR requires accurate repair of the genome in the presence of a foreign gene template.
  • Human pluripotent stem cells can be a potential source of different types of adult cells and organ bodies in vitro due to their pluripotency, and their rapid proliferation ability makes them sufficient raw materials for scientific research analysis and large-scale drug screening.
  • the application of gene editing technology in human pluripotent stem cells is of great significance to realize disease simulation and promote the development of regenerative medicine, and has become a research hotspot in the field of regenerative medicine.
  • Pluripotent Stem Cells the efficiency of HDR precise editing of the genome through Cas9 protein shearing is extremely low. Improving gene knock-in efficiency in Pluripotent Stem Cells has become a key technical issue to be resolved.
  • selection of markers to obtain monoclonal positive cell lines is usually required, and selection markers may affect cell functions, limiting their use in research and clinical applications.
  • the screening markers can be deleted through the piggyBac or Cre / loxP systems, the Cre / loxP system will retain large fragments of loxP sequences on the genome.
  • the piggyBac system requires the presence of TTAA sites nearby to prevent the introduction of other foreign sequences.
  • the purpose of the present disclosure is to achieve a technical solution that can significantly improve the efficiency of knocking in large-scale genes into stem cells.
  • the present disclosure provides a method for gene-directed knock-in in stem cells.
  • the method includes the following steps: S1. Mix sgRNA and Cas9 protein for a target site to be knocked in to form an RNP complex.
  • S2. Package the homologous recombination vector with the template DNA inserted into the AAV virus to form AAV virus particles to be transfected;
  • S3. Mix the suspension of stem cells with the suspension of the RNP complex and electrotransform to obtain electrotransformation S4.
  • the suspension of AAV virus particles to be transfected is added to the electrotransformed material to perform transfection for 4-30 hours.
  • S5. The transfected culture is subjected to limiting dilution, and then subjected to monoclonal culture, and a monoclonal cell line subjected to targeted gene knock-in is selected by PCR and sequencing.
  • the present invention establishes a technical scheme that can significantly improve the efficiency of large-scale gene knock-in into human stem cells, and can further establish the need for no screening markers to detect the knock-in of different stem cell strains when the reporter gene is knocked in The method of large fragment gene efficiency, through which the gene knock-in efficiency of different gene loci in different stem cell lines can be detected, so as to select the best scientific research and clinical application scheme.
  • the present disclosure provides a method for gene-directed knock-in in stem cells.
  • the method includes the following steps: S1, mixing sgRNA and Cas9 protein for a target site to be knocked in to form an RNP complex; S2, inserting The homologous recombination vector with template DNA is packaged into AAV virus to form AAV virus particles to be transfected; S3. A suspension of stem cells and a suspension of the RNP complex are mixed and electrotransformed to obtain an electrotransformed culture; S4. Within 1-30 minutes after the end of electrotransfection, add a suspension of AAV virus particles to be transfected to the electrotransformed material to perform transfection for 4-30 hours to obtain a transfected culture; S5 2. The said transfected culture is subjected to limiting dilution and then subjected to monoclonalization culture, and a monoclonal cell line subjected to targeted gene knock-in is selected by PCR and sequencing.
  • electroporation refers to the electrotransformation of DNA, also known as high-voltage electroproration (electroproration for short), which can be used to introduce DNA into cells.
  • the sgRNA and Cas9 protein are transferred into the cell through the RNP complex by electroporation, and the AAV virus is selected to transfect the vector inserted with the template DNA into the cell at a suitable time after the electroporation, and finally the sgRNA,
  • the Cas9 protein and the vector into which the template DNA is inserted are used by CRISPR gene editing to enable large fragments of foreign genes to be directed into the target location.
  • the sgRNA has a chemical modification group; the molar ratio of the sgRNA and Cas9 protein to the target site to be edited is 1: 1 to 1: 5.
  • an online sgRNA design tool http://crispr.mit.edu/) can be used to design and synthesize the sgRNA sequence according to the target site to be edited.
  • the 5 'end and the 3' end of the sgRNA sequence may be added with a methyl (-O-Me) chemical modification group or a -phosphorothioate chemical modification group, respectively.
  • the time for mixing the sgRNA and Cas9 protein for the target site to be edited is 5-20 minutes, and the temperature is 10-40 ° C.
  • step S3 the mixed cell suspension and the RNP complex was dry, per 106 of the stem cells to the sgRNA meter, an amount of the RNP complex 1 -50 ⁇ mol; in the suspension of the stem cells, the cell concentration was (1-5) ⁇ 10 7 cells / mL; and the final concentration of the RNP complex was 0.1-1.5 ⁇ mol / ⁇ L based on the amount of sgRNA.
  • the conditions of the electric rotation include: the electric field strength is 50-250V / cm, the single pulse time is 2-15ms, the time interval between two adjacent pulses is 10-60s, and the total pulse The number of times is 2-10 times.
  • step S4 a suspension of AAV virus particles to be transfected is added to the electrotransformed material within 5-20 minutes after the end of electrotransformation to perform transfection for 8-24 hours to obtain Transfected culture.
  • the amount of the suspension of the AAV virus particles to be transfected is such that the MOI value of the AAV virus particles to be transfected is 10 4 -10 6 .
  • the MOI value is the ratio of the virus to the number of cells at the time of infection.
  • the stem cell may be at least one of an embryonic stem cell, an induced pluripotent stem cell, and a mesenchymal stem cell; preferably, the stem cell is a human induced pluripotent stem cell 1016 cell line.
  • the serotype of the AAV virus is AAV-6 virus, AAV-1 virus, or AAV-DJ virus, and preferably the serotype of the AAV virus is AAV-DJ virus.
  • a large fragment gene knockout can be further increased Efficiency of stem cells.
  • the template DNA may include a left homology arm sequence, a knock-in fragment, and a right homology arm sequence; the left homology arm sequence and the right homology arm sequence together determine the precise insertion site, which can be determined by The site where the left homology arm sequence and the right homology arm sequence accurately control the insertion of the knock-in fragment is the target site to be knocked in.
  • the knock-in fragment includes an enhancer, a promoter, a knock-in gene, and polyA.
  • the length of the knock-in gene can be 200-5000 bp. Compared with the existing targeted knock-in method, the method of the present disclosure significantly increases the length of the knock-in gene with the same success rate. .
  • the knock-in gene is a reporter gene; in this preferred case, a screening marker can be used to detect the efficiency of knocking in large fragments of genes from different stem cell strains; further preferably, the reporter gene is A fluorescent protein reporter; the fluorescent protein reporter may be an EGFP reporter.
  • the sequence of the sgRNA of the target site to be knocked in is shown in SEQ ID NO.1; the sequence of the template DNA is shown in SEQ ID NO.6
  • the backbone sequence of the vector is shown in SEQ ID NO.7.
  • the backbone sequence of the vector refers to the sequence of the vector to which the template DNA is not inserted.
  • the present disclosure provides a second preferred embodiment.
  • the method for gene-directed knock-in in stem cells is a method for preparing a cell model capable of detecting senescence of mesenchymal stem cells in real time.
  • the method includes the following steps: S1, SgRNA and Cas9 protein targeting knock-in sites of aging-related genes are mixed to form an RNP complex; the aging-related genes include at least one of Klotho, p16, p21, p53, GATA4, SIRT1, SIRT3, SIRT6, and MORF4
  • the knock-in site of the aging-related gene is between the last sense codon and the stop codon of the aging-related gene;
  • S2 packaging the template DNA homologous recombination vector into the AAV virus to form AAV virus particle to be transfected;
  • the template DNA includes a left homology arm sequence and a right homology arm sequence directed at a knock-in site of the aging-related gene, the left homology arm sequence and the right homology arm A self-cleaved 2
  • Gene synthesis S3. Mix the suspension of mesenchymal stem cells with the suspension of the RNP complex and electrotransform to obtain the electrotransformed culture; S4. Transfer to the electrotransformation within 1-30 minutes after the electrotransformation is completed. A suspension of AAV virus particles to be transfected is added to the resulting material to perform transfection for 4-30 hours to obtain a transfected culture; S5. Monoclonal dilution of the transfected culture is performed after monoclonal dilution. Incubation, and screening of monoclonal cell lines with targeted gene knock-in by PCR and sequencing.
  • the present disclosure establishes a method for efficiently knocking in long reporter genes into mesenchymal stem cells, and inserting the reporter genes in situ without affecting the expression of aging-related genes Therefore, a variety of cell models related to the senescence of mesenchymal stem cells can be constructed, and further, during the preparation of stem cell drugs, real-time monitoring of continuous passage of cells for aging can be realized, and the prepared cells can be detected in animal models.
  • Mesenchymal stem cell drugs are transplanted to different tissues in the body, in the tissue or microenvironment of disease or aging, whether tissue-specific cytokines and harmful metabolites will affect the senescence of mesenchymal stem cells in the body and affect the survival time and function of the cells. It is convenient to conduct in-depth research on the in vivo action time and effectiveness of mesenchymal stem cells, and promote their clinical transformation and quality monitoring in industrial production.
  • the senescence gene is defined by Gene Symbol in the NCBI database.
  • the specific information is shown in Table 2-1.
  • the corresponding representative sgRNA sequences and left and right homology arms The sequences are also listed in Table 2-1.
  • the sgRNA is shown as SEQ ID No. 14, 17, 20, 23, 26, 29, 32, 35 or 38; correspondingly, the left homology arm sequence is shown as SEQ ID NO. 15, 18, 21, 24, 27, 30, 33, 36, or 39; correspondingly, the right homology arm sequence is shown in SEQ ID NO. 16, 19, 22, 25, 28, 31, 34, 37, or 40 .
  • the mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, uterine blood mesenchymal stem cells, and dental pulp.
  • Mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, uterine blood mesenchymal stem cells, and dental pulp.
  • Mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, uterine blood mesenchymal stem cells, and dental pulp.
  • Mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, umbilical cord mesenchymal stem cells, uterine blood mesenchymal stem cells, and dental pulp.
  • the self-shearing 2A peptide is used to cleave proteins and fluorescent proteins of aging-related genes, which may be at least one of a T2A peptide, an F2A peptide, and a P2A peptide
  • the fluorescent protein reporter gene may be selected widely, for example, it may be at least one of EGFP, ECFP, EYFP, ERFP, mCherry, tdTomato, Venus.
  • the sequence of the fluorescent protein reporter gene is shown in SEQ ID NO.43; the backbone sequence of the vector is shown in SEQ ID NO.44.
  • the backbone sequence of the vector refers to the sequence of the vector to which the template DNA is not inserted.
  • the present disclosure provides a third preferred embodiment. .
  • a method for gene-directed knock-in in stem cells is a method for preparing mesenchymal stem cells that can release immunosuppression and enhance tumor-targeted killing.
  • the method includes the following steps: S1.
  • sgRNA and Cas9 protein targeting the knock-in site of the PD-1 gene to form an RNP complex S2, packaging a template DNA homologous recombination vector into AAV virus to form AAV virus particles to be transfected;
  • the template DNA includes a left homology arm sequence and a right homology arm sequence for the knock-in site of the PD-1 gene, and a TRAIL gene is inserted between the left homology arm sequence and the right homology arm sequence;
  • the PD-1 gene expression can be silenced by targeted knock-in of the TRAIL gene;
  • S3 A suspension of mesenchymal stem cells and a suspension of the RNP complex are mixed and electrotransformed to obtain an electrotransformed culture; S4 1.
  • the present invention establishes a method capable of efficiently knocking in long reporter genes in mesenchymal stem cells, inserting tumor necrosis while knocking out PD-1 gene in mesenchymal stem cells TNF-related apoptosis-inducing ligand (TRAIL) gene, by knocking down PD-1 to achieve the release of CD8 + T cell inhibition (the number of cells in G0 / G1 phase decreases, and the number of cells in S phase increases)
  • TRAIL apoptosis-inducing ligand
  • a human mesenchymal stem cell capable of releasing immunosuppression and enhancing tumor targeted killing was prepared.
  • the invention also solves the problems of immunosuppression and conventional drugs with high toxicity and no targeting in the common microenvironment in tumor treatment.
  • the PD-1 gene is defined by the Gene Symbol in the NCBI database, and the NCBI Gene ID is 5133.
  • the knock-in site of the PD-1 gene can silence the expression of the PD-1 gene by targeted knock-in of the TRAIL gene, preferably the PD-1 gene.
  • the knock-in site is on the first exon of the PD-1 gene.
  • the TRAIL gene refers to a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene, and may be a membrane-bound TRAIL gene or a secretion. TRAIL gene.
  • TRAIL tumor necrosis factor-related apoptosis-inducing ligand
  • the template DNA may include a left homology arm sequence, a knock-in fragment, and a right homology arm sequence; preferably, the left homology arm sequence and the right homology arm sequence CMV enhancer, CMV promoter, TRAIL gene expression frame and SV40polyA can be inserted between the source arm sequences; preferably, the sequence inserted between the left homology arm sequence and the right homology arm sequence is as SEQ ID No. 56 or SEQ ID No. 62.
  • the sgRNA is shown as SEQ ID NO.53; correspondingly, the left homology arm sequence is shown as SEQ ID NO.54; accordingly, The right homology arm sequence is shown in SEQ ID NO.55.
  • the backbone sequence of the vector is as shown in SEQ ID NO.59.
  • the backbone sequence of the vector refers to the sequence of the vector to which the template DNA is not inserted.
  • the present disclosure provides a fourth preferred embodiment.
  • the method for gene-directed knock-in in stem cells is a method for preparing a cell model capable of detecting bone differentiation of mesenchymal stem cells in real time.
  • the method includes the following steps: S1.
  • the sgRNA and Cas9 protein of the knock-in site of bone differentiation-related genes are mixed to form an RNP complex;
  • the bone differentiation-related genes include at least one of SPP1, COL1A1, BMP-2, Runx2, SLP1, IBSP, and BGLAP;
  • the knock-in site of the bone differentiation-related gene is between the last sense codon and the stop codon of the bone differentiation-related gene;
  • S2 packaging the template DNA homologous recombination vector into the AAV virus to form AAV virus particle to be transfected;
  • the template DNA includes a left homology arm sequence and a right homology arm sequence directed at a knock-in site of the bone differentiation-related gene, the left homology arm sequence and the right homology A self-cleaved 2A peptide
  • a method for efficiently knocking in long reporter genes in mesenchymal stem cells is established, and the reporter genes are inserted in situ without affecting the expression of genes related to bone differentiation.
  • This can construct a variety of cell models related to bone differentiation of mesenchymal stem cells, and further can realize the real-time monitoring of the continuous osteoblast differentiation status of stem cell during the preparation of stem cell drugs, and the prepared mesenchymes can be detected in animal models.
  • the osteogenic differentiation gene is defined by Gene Symbol in the NCBI database.
  • the specific information is shown in Table 4-1.
  • the corresponding representative sgRNA sequence is the same as The source arm sequences are also listed in Table 4-1.
  • the sgRNA is shown as SEQ ID NO. 65, 68, 71, 74, 77, 80 or 83; correspondingly, the left homology arm sequence is shown as SEQ ID NO. 66, 69, 72, 75, 78, 81, or 84; correspondingly, the right homology arm sequence is shown in SEQ ID NO. 67, 70, 73, 76, 79, 82, or 85.
  • the self-shearing 2A peptide is used to cleave proteins and fluorescent proteins of genes related to bone differentiation, which may be at least one of a T2A peptide, an F2A peptide, and a P2A peptide.
  • the fluorescent protein reporter gene can be selected widely, for example, it can be at least one of EGFP, ECFP, EYFP, GFP, RFP, mCherry, tdTomato, and Venus.
  • the sequence of the fluorescent protein reporter gene is shown in SEQ ID NO. 88; the backbone sequence of the vector is shown in SEQ ID NO. 89.
  • the backbone sequence of the vector refers to the sequence of the vector to which the template DNA is not inserted.
  • the present disclosure provides a fifth preferred embodiment .
  • a method for gene-directed knock-in in stem cells is a method for preparing a cell model capable of detecting real-time differentiation of mesenchymal stem cells into mature liver-like cells.
  • the method includes the following: Steps: S1, mixing sgRNA and Cas9 protein targeting knock-in sites of liver-like cell-related genes to form an RNP complex; the liver-like cell-related genes include ALB and / or AFP; The knock-in site is between the last sense codon and the stop codon of the liver-like cell-related gene; S2, packaging the template DNA homologous recombination vector into the AAV virus to form the AAV virus to be transfected Particles; the template DNA includes a left homology arm sequence and a right homology arm sequence for the knock-in site of the liver-like cell-related gene, between the left homology arm sequence and the right homology arm sequence A self-shearing 2A peptide coding sequence and a fluorescent protein reporter gene are inserted
  • the suspension of the mesenchymal stem cells is mixed with the suspension of the RNP complex and electrotransformed to obtain the electrotransformed culture; S4. Within 1-30 minutes after the electrotransformation is completed, the electrotransformed material is sent to the electrotransformed material. A suspension of AAV virus particles to be transfected is added to the transfection for 4-30 hours to obtain a transfected culture; S5. The transfected culture is diluted and subjected to monoclonal culture, Monoclonal cell lines were screened by PCR and sequencing.
  • the present disclosure establishes a method capable of efficiently knocking in long reporter genes in mesenchymal stem cells without affecting the liver differentiation and maturity marker ALB gene and liver immature standards.
  • the reporter gene is inserted in situ, thereby constructing a cell model that can monitor the differentiation and maturation of differentiated mesenchymal stem cells to mature liver-like cells in real time during the preparation of stem cell drugs.
  • the liver-like cell-related genes are defined by Gene Symbol in the NCBI database, and specific information is shown in Table 5-1.
  • the corresponding representative sgRNA The sequences and left and right homology arm sequences are also listed in Table 5-1.
  • the sgRNA is shown as SEQ ID NO. 98 or 101; correspondingly, the left homology arm sequence is shown as SEQ ID NO. 99 or 102; correspondingly, the right homology arm sequence is shown as SEQ ID No. 100 or 103.
  • the self-shearing 2A peptide is used to cleave proteins and fluorescent proteins of liver-like cell-related genes, which may be at least one of T2A peptide, F2A peptide, and P2A peptide; selection of the fluorescent protein reporter gene It can be wider, for example, it can be at least one of EGFP, ECFP, EYFP, GFP, RFP, mCherry, tdTomato, and Venus.
  • the sequence of the fluorescent protein reporter gene is shown in SEQ ID NO. 106; the backbone sequence of the vector is shown in SEQ ID NO. 10107.
  • the backbone sequence of the vector refers to the sequence of the vector to which the template DNA is not inserted.
  • the present disclosure provides a sixth preferred embodiment.
  • the present disclosure provides a method for preparing mesenchymal stem cells that resists cellular senescence and prolongs the time course of blood glucose regulation.
  • the method includes the following steps: S1. Inserting a target gene into a safety island The sgRNA and Cas9 protein of the knock-in site are mixed to form an RNP complex; S2, the template DNA homologous recombination vector is packaged into AAV virus to form AAV virus particles to be transfected; the template DNA includes a gene insertion The left homology arm sequence and the right homology arm sequence at the knock-in site of the safety island, and the GPx7 gene is inserted between the left homology arm sequence and the right homology arm sequence; S3.
  • the iPSC cells or embryonic stem cells The suspension is mixed with the suspension of the RNP complex and electrotransformed to obtain the electrotransformed culture; S4. Within 1-30 minutes after the electrotransformation is completed, the AAV virus to be transfected is added to the electrotransformed material. The suspension of the particles is transfected for 4-30 hours to obtain a transfected culture; S5. The transfected culture is subjected to limiting dilution and then subjected to monoclonal culture, and selected by PCR and sequencing. Gene knock-directed monoclonal cell line; S6, the orientation of the gene was knockin cell clone was amplified and induced to differentiate into mesenchymal stem cells.
  • the present disclosure also provides mesenchymal stem cells prepared by the above method.
  • the present disclosure also provides the use of the above-mentioned mesenchymal stem cells, which is any of the following: preparing a product that improves insulin resistance and / or chronic inflammation; preparing a cell transplantation therapy Products; preparation of therapeutic products to improve beta cell function; preparation of products to regenerate and / or repair damaged blood vessels.
  • the present disclosure accurately and efficiently inserts a GPx7 expression element into a safe island (including rDNA28s, rDNA, 18S, rDNA45s, CLYBL, and AAVS1) at a gene insertion site of mesenchymal stem cells to increase the expression of GPx7.
  • a safe island including rDNA28s, rDNA, 18S, rDNA45s, CLYBL, and AAVS1
  • mesenchymal stem cells to increase the expression of GPx7.
  • the gene insertion safety island is defined as a safe insertion site of a foreign gene, and the insertion of the foreign gene does not affect the gene expression of the cell itself;
  • the gene insertion into the safety island Can be RNA45SN4, rDNA 28S (NCBI Gene ID 106632264), rDNA 18S (NCBI Gene ID 106631781), rDNA 45S (NCBI Gene ID 109864271), CLYBL (NCBI Gene ID 171425), or AAVS1 (NCBI Gene ID 17) );
  • the inserted gene GPx7 has an NCBI Gene ID of 2882.
  • the gene insertion safety island is RNA45SN4, and its NCBI Gene ID is 109864271.
  • the GPx7 gene refers to an endoplasmic reticulum glutathione peroxidase 7 gene, and its NCBI Gene ID is 2882.
  • the template DNA may include a left homology arm sequence, a knock-in fragment, and a right homology arm sequence; preferably, the left homology arm sequence and the right homology arm sequence A CMV enhancer, a CMV promoter, an expression frame of the GPx7 gene, and bGH polyA may be inserted between the homology arm sequences; preferably, a sequence inserted between the left homology arm sequence and the right homology arm sequence As shown in SEQ ID NO.117.
  • the sgRNA is shown as SEQ ID NO. 114; correspondingly, the left homology arm sequence is shown as SEQ ID NO. 115; accordingly The sequence of the right homology arm is shown in SEQ ID NO.116.
  • the backbone sequence of the vector is as shown in SEQ ID NO. 120.
  • the backbone sequence of the vector refers to the sequence of the vector to which the template DNA is not inserted.
  • the conditions for expanding and directing induced differentiation include: continuing the genetically-directed knock-in monoclonal cell strain in a low-adhesion culture plate After 18-100 hours of culture, embryoid bodies are formed; then the embryoid bodies are seeded on matrigel and cultured for 10-20 days, and then a cell population in which CD73, CD90 and CD105 are all positive is sorted by flow cytometry.
  • Example 1-1 is used to describe the first preferred embodiment of the present disclosure in detail.
  • a targeted HBB sgRNA was designed on the exon 1 of the HBB gene by an online sgRNA design tool (http://crispr.mit.edu/).
  • HBB sgRNA 5'-cuugccccacagggcaguaaguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuuuuu-3 '(SEQ ID ID NO. 1).
  • sequences of positions 1-20 in HBB sgRNA are recognition motifs, and the rest are tracrRNA.
  • HBB sgRNA was synthesized from Integrated DNA Technologies and added with O-Me and phosphorothioate modifications at the 5 'and 3' ends of the HBB sgRNA sequence, respectively.
  • HBB-FW 5’-tagatgtccccagttaacctcctat-3 ’(SEQ ID NO. 2),
  • HBB-REV 5'-ttattaggcagaatccagatgctca-3 '(SEQ ID NO. 3).
  • the cell to be edited is a human induced pluripotent stem cell 1016 cell line.
  • a packaging system with a serotype of AAV-DJ is preferred.
  • the total packaging capacity of AAV-DJ is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO. 4, in which the 495th to 497th bases are mutated PAM sites.
  • the sequence of the right homology arm is SEQ ID NO.5.
  • the insert is a complete Cassette, including CMV increaser, CMV promoter, EGFP and bGH poly A, a total of about 1452bp, as shown in SEQ ID NO. 6, where the 1-507th is the promoter, the 508-1227th EGFP, bGH polyA at positions 1228-1452.
  • Cashette was inserted into the pAAV vector using NheI and BsmI restriction enzymes (purchased from NEB (Beijing) Co., Ltd.), and the vector sequence was SEQ ID NO.7.
  • PrimerStar high-fidelity DNA polymerase purchased from Bao Biotechnology (Dalian) Co., Ltd., article number: R044A was used to engrav the loop.
  • the method introduced a point mutation (CGG to CTG) at the PAM site, thereby avoiding the cleavage of the recognition site by the SpCas9 / sgRNA and RNP complex.
  • Point mutation primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • HBB-PAM-mut-FW 5'-cctgaggagaagtctgcagttactgccctgtgggg-3 '(SEQ ID NO. 8).
  • HBB-PAM-mut-REV 5'-ccccacagggcagtaactgcagacttctcctcagg-3 '(SEQ ID NO. 9).
  • HEK293T cells were seeded in a quantity of 5 ⁇ 10 6 per dish to a 10 cm diameter containing 10 mL of complete medium (DMEM + 10% FBS + 1% P / S double antibody) (DMEM medium, CORNING petri dish purchased from ThermoFisher Scientific, Inc., article number: C11995500BT; FBS, purchased from ThermoFisher Scientific, Inc., article number: sv30087.02; P / S dual antibody, purchased from ThermoFisher Scientific, Inc., article number: SV30010) In total, 30 dishes were planted. The cells were cultured in a 37 ° C, 5% CO 2 cell incubator for 24 hours.
  • complete medium DMEM + 10% FBS + 1% P / S double antibody
  • the collected supernatant was centrifuged at 4000 rpm and 4 degrees for 10 minutes, and then the impurities were discarded.
  • the impurity-removed supernatant was added to an Amicon Ultra-15 ultra-column column (purchased from Merck Chemicals (Shanghai) Co., Ltd., article number: UFC905096), and the volume was concentrated to 10 to 10 to 4,000 rpm and 4 degrees centrifugation for 30 minutes. 15mL.
  • HEK293T cells scraped with a cell spatula were blown with an appropriate amount of medium and transferred to a 50 mL centrifuge tube. After centrifugation at 1500 rpm and 4 degrees for 10 min, the supernatant was discarded.
  • a total of 3 mL of cell lysis buffer (150 mM NaCl, 20 mM tritris) was added to all the pellets. (pH 8.0).
  • the resuspended cells were repeatedly freeze-thawed three times in a -80 ° C alcohol bath and a 37 ° C water bath. Mix the concentrated supernatant and the freeze-thaw cell suspension, add 1M MgCl2 to a final concentration of 1mM.
  • Add Benzonase (purchased from Merck Chemicals (Shanghai) Co., Ltd., article number: 70746-1kU) to a final concentration of 25U / mL, and mix for 40 minutes at 37 ° C after mixing. Take out the 50mL centrifuge tube, centrifuge at 4 °C, 4000rpm for 20min, and take the supernatant.
  • the virus was purified by iodixanol density gradient centrifugation (purchased from Sigma-Aldrich, article number: D1556-250mL).
  • Configure iodixanol gradient 17%: 5mL 10 ⁇ PBS, 0.05mL 1M MgCl 2 , 0.125mL 1M KCl, 10mL 5M NaCl, 12.5mL Optiprep, and make up to 50mL with water.
  • 25% 5mL 10 ⁇ PBS, 0.05mL 1M MgCl2, 0.125mL 1M KCl, 20mL Optiprep, 0.1mL 0.5% phenol red, make up to 50mL with water.
  • Beckman L-80XP floor ultracentrifuge 70Ti fixed-angle rotor, acceleration 6, deceleration 9,600,000 rpm, 4 degrees centrifugation for 2 hours. Pipet a 40% concentration layer of iodixanol with a flat-tip syringe, transfer to an Amicon Ultra-15 column, add 10 mL of PBS at 4000 rpm and centrifuge at 4 ° C for 20 minutes, and repeat 3 times. The virus was concentrated by centrifugation to 1 mL.
  • the titer of AAV-DJ was detected by qPCR.
  • the primers were designed based on the EGFP sequence so that the length of the qPCR product was about 200bp.
  • qPCR primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • AAVGFPF 5'-tcagcttcaggcaccaccac-3 '(SEQ ID NO. 10).
  • AAVGFPR 5'-tgaacttgtggccgtttacgtcg-3 '(SEQ ID NO. 11).
  • AAV-DJ recombinant plasmid standards were prepared and diluted 1:10. Dilution starts from concentrations below 10ng / ⁇ L, which are 10ng / ⁇ L, 1ng / ⁇ L, 0.1ng / ⁇ L, 0.01ng / ⁇ L, 0.001ng / ⁇ L, 0.0001ng / ⁇ L, and 0.00001ng / ⁇ L, respectively.
  • Virus samples were pretreated using DNaseI (purchased from Bao Bioengineering (Dalian) Co., Ltd., article number: 2270A).
  • DNaseI purchased from Bao Bioengineering (Dalian) Co., Ltd., article number: 2270A
  • a 2 ⁇ SYBR PCR mix purchased from Toyobo (Shanghai) Biotechnology Co., Ltd., QPS-201 was used to configure the qPCR reaction system.
  • Roche 480II real-time PCR system for quantitative PCR. Based on the Ct value, a standard curve was drawn and the titer of AAV-DJ was calculated.
  • SpCas9 final concentration 1 ⁇ mol / ⁇ L
  • HBB gsgRNA final concentration 1 ⁇ mol / ⁇ L
  • Opti-MEM (14.5 mM ATP, 23.6 mM magnesium chloride) (commercially available from ThermoFisher Scientific, Inc., Cat. No. 11058021).
  • cells were transferred to geltrex-coated (purchased from ThermoFisher Scientific, Inc., article number: A1413202) and added with 500 ⁇ L of mTeSR and 10 ⁇ M Y-27632 (purchased from Stem Cell Technologies, Inc., item: 72304). The culture was continued in a 24-well plate in a 37 ° C, 5% CO 2 cell incubator.
  • AAV-DJ is gently added at a MOI value of 1 ⁇ 10 5 within 5 to 20 minutes after the electric transfer, and the addition is completed within 20 minutes after the electric transfer.
  • Cells were seeded at a rate of 15000 cells per 10cm dish into a 10cm CORNING dish coated with Geltrex and added with 10mL mTeSR and 10 ⁇ M Y-27632 in a 37 ° C, 5% CO 2 cell culture incubator. .
  • the size of the clone is equivalent to a coin under a ten-fold objective lens 12 to 14 days after the electric transfer. Do not allow the clones to continue to grow or intersect.
  • 120 ⁇ L mTeSR (containing 10 ⁇ M Y-27632) was added to each well of a 96-well plate coated with Geltrex, and plate O was labeled. Observe through a microscope in a clean bench, adjust the P200 pipette to 45 ⁇ L, scrape the clones with a pipette tip with a filter element, collect the cells with a pipette, and transfer to the wells of a 96-well plate.
  • HBB-FW 5’-tagatgtccccagttaacctcctat-3 ’(SEQ ID NO.12),
  • HBB-REV 5'-ttattaggcagaatccagatgctca-3 '(SEQ ID NO. 13).
  • PCR products are electrophoresed and analyzed. Only 2400bp bands are non-editing cells, only 3900bp bands are bi-allelic editing cells, and both 2400bp bands and 3900bp bands are single-allele editing cells. Mark and count the proportions of these three types of editing types separately.
  • Example 1-1 The method of Example 1-1 was followed, except that the AAV-DJ was gently added at a MOI of 1 ⁇ 10 5 at the 35th minute after the electric transfer, and the addition was completed within 50 minutes after the electric transfer.
  • Test sample on the fourth day after electroporation and AAV infection, after passing Accutase digestion enzyme, fresh mTeSR medium was added and the stem cell suspension was resuspended.
  • Detection method Because Cassette inserted at the target locus can express green fluorescent protein, use a flow cytometer to detect and count the number of cells that can emit green fluorescence and the total number of cells. Dividing the number of green fluorescent cells by the total number of cells gives you the total editing efficiency.
  • Example 1-1 The editing efficiency of Example 1-1 is 81.2%, and the editing efficiency of Comparative Example 1-1 is 10.6%.
  • Test samples monoclonal cell lines (plates A and B) cultured in 96-well plates
  • Detection method Design primers outside the homology arms (non-integrated site PCR product is about 2400bp, integrated site PCR product is 4600bp). The primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • Example 1-1 and Comparative Example 1-1 used HBB-FW: 5'-tagatgtccccagttaacctcctat-3 '(SEQ ID NO. 12)
  • HBB-REV 5'-ttaggcagaatccagatgctca-3 '(SEQ ID NO. 13).
  • the genome obtained from the clones in the 96-well plate was subjected to PCR amplification.
  • the PCR products were electrophoresed and analyzed. Only 2400bp bands are non-editing cells, only 4600bp bands are biallele-editing cells, and both 2400bp bands and 4600bp bands are single allele-editing cells.
  • Example 1-1 editing efficiency allele double editing 64.2%, allele single editing 16.4%
  • comparative example 1-1 editing efficiency allele double editing 0.3%
  • allele single editing 0.7% It can be seen that the time interval between electrotransfection and viral transfection has a greater impact on the efficiency of gene knock-in. It is preferred to add a suspension of AAV virus particles to be transfected to the electrotransformed material within 5-20 minutes after the end of electrotransfection In the case of transfection for 8-24 hours, the efficiency of gene knock-in is the highest.
  • Example 2-1 is used to describe a second preferred embodiment of the present disclosure in detail.
  • a cell model was constructed by inserting the EGFP reporter gene into the Klotho gene of mesenchymal stem cells.
  • Klotho gene in situ promoter was used, and T2A-EGFP was inserted before the stop codon.
  • sequences at positions 1-20 are recognition motifs, and the rest are tracrRNA.
  • the sgRNA was synthesized by Integrated DNA Technologies, and O-Me and phosphorothioate were added to the 5 'and 3' ends of the sgRNA sequence, respectively.
  • the amplified target sequence fragment (2786bp) was amplified from the genome, and the primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • Klotho-REV 5'-gtatgtcattaaccagatacat-3 '(SEQ ID NO. 42).
  • the cells to be edited are human bone marrow mesenchymal stem cells. According to the AAV tissue affinity control table, a packaging system of serotype AAV-9 is preferred.
  • the total packaging capacity of AAV-9 is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO. 15, wherein the 412-414th base is the mutated PAM site.
  • the sequence of the right homology arm is SEQ ID NO.16.
  • the inserts are T2A and EGFP, a total of 777bp, and the sequence is shown in SEQ ID NO.43.
  • Cassette was inserted into the pAAV vector using the method of seamless cloning (NEBuilder high-fidelity DNA assembly kit, purchased from NEB (Beijing) Co., Ltd., E2621S), and the vector sequence is SEQ ID NO.44.
  • PrimerStar high-fidelity DNA polymerase purchased from Bao Biotechnology (Dalian) Co., Ltd., article number: R044A
  • the method introduced a point mutation (TGG to TCG) at the PAM site, thereby avoiding the cleavage of the recognition site by the SpCas9 / sgRNA and RNP complex.
  • Point mutation primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • Klotho-PAM-mut-FW 5'-acccgaaagtctttactcgctttcatagcttttct-3 '(SEQ ID NO. 45).
  • Klotho-PAM-mut-REV 5'-agaaaagctatgaaagcgagtaagactttcgggt (SEQ ID NO. 46).
  • the HEK293T cells were seeded one day to 10cm in diameter by the number of 5 ⁇ 10 6 per dish in front of the package containing the virus 10mL complete medium (DMEM (commercially available from ThermoFisher Scientific, Inc, NO:. C11995500BT) + 10% FBS ( Purchased from ThermoFisher Scientific, Inc., article number: SV30087.02) + 1% PS double antibody was purchased from ThermoFisher Scientific, Inc., article number: SV30010), and a total of 30 dishes were planted. The cells were cultured in a 37 ° C, 5% CO 2 cell incubator for 24 hours.
  • DMEM virus 10mL complete medium
  • FBS Purchased from ThermoFisher Scientific, Inc., article number: SV30087.02
  • PS double antibody was purchased from ThermoFisher Scientific, Inc., article number: SV30010
  • the collected supernatant was centrifuged at 4000 rpm at 4 degrees for 10 minutes, and the impurities were discarded.
  • the impurity-removed supernatant was added to an Amicon Ultra-15 column (purchased from Merck Chemical Technology (Shanghai) Co., Ltd., article number: UFC 905096). After several centrifugations at 4000 rpm and 4 degrees for 30 minutes, the volume was concentrated to 10 to 15mL.
  • HEK293T cells scraped with a cell spatula were blown with an appropriate amount of medium and transferred to a 50 mL centrifuge tube. After centrifugation at 1500 rpm and 4 degrees for 10 min, the supernatant was discarded.
  • a total of 3 mL of cell lysis buffer (150 mM NaCl, 20 mM tris was added to all the pellets. (pH 8.0).
  • the resuspended cells were repeatedly freeze-thawed three times in a -80 ° C alcohol bath and a 37 ° C water bath.
  • the concentrated supernatant and the freeze-thawed cell suspension were mixed and 1 M MgCl 2 was added to a final concentration of 1 mM.
  • Add Benzonase (purchased from Merck Chemicals (Shanghai) Co., Ltd., article number: 70746-1kU) to a final concentration of 25U / mL, and mix for 40 minutes at 37 ° C after mixing. Take out the 50mL centrifuge tube, centrifuge at 4 °C, 4000rpm for 20min, and take the supernatant.
  • Virus was purified by iodixanol density gradient centrifugation as described above.
  • AAV9 was tested for titer by qPCR.
  • the primers were designed based on the EGFP sequence so that the length of the qPCR product was about 200bp.
  • qPCR primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • AAVGFPF 5'-tcagcttcaggcaccaccac-3 '(SEQ ID NO. 47).
  • AAVGFPR 5'-tgaacttgtggccgtttacgtcg-3 '(SEQ ID NO. 48).
  • SpCas9 final concentration: 300 ug / ml
  • Klotho sgRNA final concentration: 175 ug / ml
  • the size of the clone is equivalent to a coin under a ten-fold objective lens 12 to 14 days after the electric transfer. Do not allow the clones to continue to grow or intersect.
  • Add 120 ⁇ L of DMEM / F12 complete medium to each well of a 96-well plate and label plate O. Observe through a microscope in a clean bench, adjust the P200 pipette to 45 ⁇ L, scrape the clones with a pipette tip with a filter element, collect the cells with a pipette, and transfer to the wells of a 96-well plate.
  • Example 2-2 is used to describe a second preferred embodiment of the present disclosure in detail.
  • a cell model was constructed by inserting the EGFP reporter gene into the bone marrow mesenchymal stem cell cycle regulatory gene p16 gene. Using the p16 gene in situ promoter, 2A-EGFP was inserted before the stop codon.
  • p16sgRNA 5'-gggccgucugcccguggaccguuuuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuuu-3 '. (SEQ ID NO.17).
  • sequences at positions 1-20 are recognition motifs, and the rest are tracrRNA.
  • the sgRNA was commissioned by Integrated DNA Technologies to synthesize, and O-methyl and -phosphorothioate modifications were made to the 5 'and 3' ends of the sgRNA, respectively.
  • P16-REV 5'-agggtcagcgaagtcttggt-3 ', (SEQ ID NO. 50).
  • the cells to be edited are adipose-derived mesenchymal (ADSC) stem cell lines.
  • ADSC adipose-derived mesenchymal stem cell lines.
  • AAV tissue affinity control table a packaging system with a serotype of AAV9 is preferred.
  • the total packaging capacity of AAV9 is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO. 18, in which the 489th to 494th bases are the second and third codons mutated upstream of the PAM site, because the PAM site cannot fail by synonymous mutation.
  • the sequence of the right homology arm is SEQ ID NO.19.
  • the inserts are T2A and EGFP, a total of 777bp, and the sequence is shown in SEQ ID NO.43.
  • Cassette was inserted into the pAAV vector using the method of seamless cloning (NEBuilder high-fidelity DNA assembly kit, purchased from NEB (Beijing) Co., Ltd., E2621S), and the vector sequence is SEQ ID NO.44.
  • P16-PAM-mut-FW 5'-cgcgatgcctggggccgtctgcccgcggccctggc-3 '(SEQ ID NO.51).
  • P16-PAM-mut-REV 5'-gccagggccgcgggcagacggccccaggcatcgcg-3 '(SEQ ID NO.52).
  • the method was carried out according to the method of Example 2-1, except that at the 35th minute after the electric transfer, AAV9 was gently added dropwise according to a MOI of 1 ⁇ 10 5 and the addition was completed within 50 minutes after the electric transfer.
  • Test samples Examples 2-1, 2-2, and Comparative Example 2-1 After electroporation and AAV infection on the fourth day after trypsin digestion, fresh DMEM / F12 complete medium was added to resuspend the stem cell suspension. liquid.
  • T2A-EGFP sequence inserted at the target locus expresses green fluorescent protein
  • use a flow cytometer to detect and count the number of cells that can emit green fluorescence and the total number of cells. Dividing the number of cells that can emit green fluorescence by the total number of cells gives you the total editing efficiency.
  • Example 2-1 The editing efficiency of Example 2-1 is 73%, the editing efficiency of Example 2-2 is 76%, and the editing efficiency of Comparative Example 2-1 is 8%.
  • Test samples monoclonal cell lines (plates A and B) cultured in 96-well plates
  • Detection method Design primers outside the homology arms.
  • the primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • Example 2-1 For Example 2-1 and Comparative Example 2-1, Klotho-FW and Klotho-REV were used. In Example 2-2, p16-FW and p16-REV were used.
  • Example 2-1 and Comparative Example 2-1 only 2786bp bands were non-editing cells, only 3563bp bands were double allele editing cells, and both 2786bp bands and 3563bp bands were single allele editing cells.
  • Example 2-2 only the band of about 1413bp is a non-editing cell, only 2190bp is a biallele-editing cell, and both the band of about 1413bp and 2190bp are a single allele-editing cell.
  • Example 2-1 editing efficiency 62% allele double editing, 77% allele single editing
  • comparative 2-1 editing efficiency 1% allele double editing
  • Example 2-2 Editing efficiency 58% allele double editing and 76% allele single editing; it can be seen that the time interval between electroporation and viral transfection has a greater effect on the efficiency of gene knock-in, and it is preferred that after the electroporation is completed In 5-20 minutes, when the suspension of AAV virus particles to be transfected is added to the electrotransformed material to perform transfection for 8-24 hours, the efficiency of gene knock-in is the highest.
  • Bone marrow mesenchymal stem cells with EGFP knock-in bone marrow mesenchymal stem cells obtained in Example 2-1 were seeded on 6-well plates, and adhered, and a DNA damage inducer with a final concentration of 100 ⁇ M was added 4- Nitroquinoline-N-oxide (purchased from Sigma-Aldrich, article number: N8141) and endoplasmic reticulum stress inducer, tunicamycin (purchased from Sigma-Aldrich, article number: 654380) at a final concentration of 6 ⁇ g / mL -10MG), with a total volume of 2 mL, was treated in two groups.
  • 4- Nitroquinoline-N-oxide purchased from Sigma-Aldrich, article number: N8141
  • tunicamycin purchased from Sigma-Aldrich, article number: 654380
  • one group After collecting and processing 0h, 12h, 24h, 48h, and 72h in sequence, one group detected the luminescence of EGFP in the sample by flow cytometry, and counted the percentage of luminescent cells; the other group was stained with ⁇ -galactosidase kit (Purchased from Beijing Solibao Technology Co., Ltd., article number: G1580) for staining, and then observed the color development of X-gal under a light microscope to calculate the percentage of color-developed cells. The results are shown in Table 2-2 (4-nitroquinoline-N-oxide) and Table 2-3 (tunicamycin).
  • Example 2-1 The above Table 2-2 and Table 2-3 show that the model established in Example 2-1 can detect cell aging under different pressures in real time without causing cell death. A decrease in the ratio of fluorescently expressed cells represents an increase in the number of senescent cells. Compared with the X-gal staining method, it has a consistent trend of cell senescence.
  • Example 2-2 The operation of Example 2-2 is the same as that of Example 2-1.
  • the luminescence or color development percentage of cells treated with 4-nitroquinoline-N-oxide or tunicamycin is shown in Table 2-4 and 2-5.
  • Example 2-2 The results in Tables 2-4 and 2-5 above show that the model established in Example 2-2 can detect cell aging under different pressures in real time without causing cell death.
  • the up-regulation of p16 gene is related to cell senescence.
  • EGFP An increase in the ratio of fluorescently expressed cells represents an increase in the number of senescent cells. Compared with the X-gal staining method, it has a consistent trend of cell senescence.
  • the bone marrow mesenchymal stem cells with EGFP knock-in bone marrow mesenchymal stem cells obtained in Example 1 were seeded in 10 cm normal dish and passaged in order.
  • the 0th, 2nd, 5th, 10th, and 15th generation cells were sequentially plated Seeded in 96 wells, detected by CellTiter kit (purchased from Promega, article number: G3582), the doubling viability of the corresponding algebraic cells was counted to characterize the senescence of the cells.
  • the cells of the corresponding generation were used to detect the luminescence of EGFP by flow cytometry, and the percentage of luminous cells was counted. The results are shown in Table 2-6.
  • Example 2-2 The operation of Example 2-2 is the same as that of Example 2-1, and the results are shown in Table 2-7.
  • Example 3-1 is used to describe a third preferred embodiment of the present disclosure in detail.
  • a cell model was constructed in which membrane-bound TRAIL gene expression Cassette was inserted into Exon1 of umbilical cord mesenchymal stem cells' programmed death receptor 1 gene (PD-1).
  • PD-1 programmed death receptor 1 gene
  • a PD-1sgRNA targeted for recognition was designed on the exon 1 of PD-1 gene by an online sgRNA design tool (http://crispr.mit.edu/). The preferred results are as follows: 5’-
  • sequences at positions 1-20 are recognition motifs, and the rest are tracrRNA.
  • the sgRNA was synthesized from Integrated DNA Technologies, Inc. (IDT) and added to the second and third positions of the three bases at the 5 'and 3' ends of the sgRNA sequence. Modification of phosphorothioate.
  • PD-1-REV 5'-agtcgcctgccacagtgaag-3 '(SEQ ID NO. 58).
  • the cells to be edited are umbilical cord mesenchymal stem cells. According to the AAV tissue affinity control table, a packaging system of serotype AAV-9 is preferred.
  • the total packaging capacity of AAV-9 is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO.54.
  • the sequence of the right homology arm is SEQ ID NO.55.
  • Cassette was inserted into the pAAV vector using the method of seamless cloning (NEBuilder high-fidelity DNA assembly kit, purchased from NEB (Beijing) Co., Ltd., E2621S), and the vector sequence is SEQ ID NO.59.
  • HEK293T cells were seeded in a quantity of 5 ⁇ 10 6 per dish to a 10 cm diameter containing 10 mL of complete medium: DMEM (purchased from ThermoFisher Scientific, Inc., article number: C11995500BT) + 10% FBS ( A total of 30 dishes were grown in a CORNING culture dish purchased from ThermoFisher Scientific, Inc., article number: sv30087.02) + 1% P / S double antibody (purchased from ThermoFisher Scientific, Inc., article number: SV30010). The cells were cultured in a 37 ° C, 5% CO 2 cell incubator for 24 hours.
  • DMEM purchased from ThermoFisher Scientific, Inc., article number: C11995500BT
  • FBS A total of 30 dishes were grown in a CORNING culture dish purchased from ThermoFisher Scientific, Inc., article number: sv30087.02
  • P / S double antibody purchased from ThermoFis
  • the collected supernatant was centrifuged at 4000 rpm at 4 degrees for 10 minutes, and the impurities were discarded.
  • the impurity-removed supernatant was added to an Amicon Ultra-15 column (purchased from Merck Chemical Technology (Shanghai) Co., Ltd., article number: UFC 905096). After several centrifugations at 4000 rpm and 4 degrees for 30 minutes, the volume was concentrated to 10 to 15mL.
  • HEK293T cells scraped with a cell spatula were blown with an appropriate amount of medium and transferred to a 50 mL centrifuge tube. After centrifugation at 1500 rpm and 4 degrees for 10 min, the supernatant was discarded.
  • a total of 3 mL of cell lysis buffer (150 mM NaCl, 20 mM tris was added to all the pellets. (pH 8.0).
  • the resuspended cells were repeatedly freeze-thawed three times in -80 ° C alcohol bath and 37 ° C water bath.
  • the concentrated supernatant and the freeze-thawed cell suspension were mixed and 1 M MgCl 2 was added to a final concentration of 1 mM.
  • Add Benzonase (purchased from Merck Chemicals (Shanghai) Co., Ltd., article number: 70746-1kU) to a final concentration of 25U / mL, and mix for 40 minutes at 37 ° C after mixing. Take out the 50mL centrifuge tube, centrifuge at 4 °C, 4000rpm for 20min, and take the supernatant.
  • the virus was purified by iodixanol density gradient centrifugation.
  • AAV9 was tested for titer by qPCR.
  • the primers were designed based on the EGFP sequence so that the length of the qPCR product was about 200bp.
  • qPCR primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • AAV-TRAIL-F 5'-gatcttcacagtgctcctgc-3 '(SEQ ID NO. 60).
  • AAV-TRAIL-R 5'-tgacggagttgccacttgac-3 '(SEQ ID NO. 61).
  • SpCas9 final concentration: 300 ug / ml
  • PD1sgRNA final concentration: 175 ⁇ g / ml
  • a stem cell suspension is prepared. Cell density was counted using a hemocytometer. The cell density was adjusted to 5 ⁇ 10 7 / mL using Opti-MEM (14.5 mM ATP, 23.6 mM magnesium chloride, purchased from ThermoFisher Scientific, Inc., Cat. No. 11058021).
  • the size of the clone is equivalent to a coin under a ten-fold objective lens 12 to 14 days after the electric transfer. Do not allow the clones to continue to grow or intersect.
  • Add 120 ⁇ L of DMEM / F12 complete medium to each well of a 96-well plate and label plate O. Observe through a microscope in a clean bench, adjust the P200 pipette to 45 ⁇ L, scrape the clones with a pipette tip with a filter element, collect the cells with a pipette, and transfer to the wells of a 96-well plate.
  • Example 3-2 is used to describe a third preferred embodiment of the present disclosure.
  • a cell model was constructed in which a secreted TRAIL gene was expressed in Exon1 of umbilical cord mesenchymal stem cells and the programmed death receptor 1 gene (PD-1).
  • Example 3-1 The method of Example 3-1 was used, except that the inserted fragment was a complete Cassette, including CMV increaser, CMV promoter, recombinant secretory TRAIL gene CDS and SV40polyA, a total of 1349bp, and the sequence is as shown in SEQ ID ID NO.62 Show.
  • Example 3-1 The method of Example 3-1 was performed, except that at the 35th minute after the electric transfer, AAV9 was gently added dropwise according to a MOI of 1 ⁇ 10 5 and the addition was completed within 50 minutes after the electric transfer.
  • Example 3-2 The method of Example 3-2 was followed, except that at the 35th minute after the electrical transfer, AAV9 was gently added dropwise according to a MOI of 1 ⁇ 10 5 and the addition was completed within 50 minutes of the electrical transfer.
  • PD1-HR-REV 5'-agaagaactgtcctcactcg-3 '(SEQ ID NO. 64).
  • the cell cycle checkpoint is a set of check mechanisms that guarantee the quality of DNA replication and chromosome allocation in the cell cycle. It is a type of negative feedback regulation mechanism. When abnormal events occur during the cell cycle, such as DNA damage or blocked DNA replication, these regulatory mechanisms are activated, interrupting the cell cycle in a timely manner. After the cells repair or troubleshoot, the cell cycle can resume operation.
  • the umbilical cord mesenchymal stem cells obtained in Examples 3-1 and 3-2 and the umbilical cord mesenchymal stem cells that were not knocked in were transplanted into BALB / c mice, respectively.
  • a human breast cancer (EAC) tumor-bearing Kunming mouse model (body weight 18-22 g) was prepared, and Examples 3-1 and 3-2 were used.
  • EAC human breast cancer
  • Examples 3-1 and 3-2 were used.
  • the obtained umbilical cord mesenchymal stem cells and umbilical cord mesenchymal stem cells without knock-in were injected into the tumor-bearing mice by tail vein injection (injection dose is 1 ⁇ 10 6 cells / 0.1ml), and the tumor suppressive effect was checked. The results are shown in the table.
  • the umbilical cord mesenchymal stem cells obtained in Examples 3-1 and 3-2 can significantly improve the tumor suppression efficiency compared with the umbilical cord mesenchymal stem cells that have not been knocked in: (average tumor weight of the tumor control group- Experimental group average tumor weight) / tumor control group average tumor weight ⁇ 100%.
  • Example 4-1 is used to describe a fourth preferred embodiment of the present disclosure in detail.
  • a cell model in which the EGFP reporter gene was inserted into the osteopontin gene (SPP1) of adipose mesenchymal stem cells (ADSC cells) was constructed.
  • SPP1 gene in situ promoter T2A-EGFP was inserted before the stop codon.
  • SPP1sgRNA 5'-gguuguagaccccaaaaguaguuuuuugagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuuuuu-3 '(SEQ ID ID NO. 65).
  • the sequences at positions 1-20 are recognition motifs, and the rest are tracrRNA.
  • the sgRNA was synthesized by Integrated Technologies and USA, and modified by adding O-Me and phosphorothioate to the second and third positions of the three bases at the 5 'end and the 3' end of the sgRNA sequence, respectively. .
  • Genomic amplification recognizes the target sequence fragment (1144bp), and the primers are synthesized by Bioengineering Engineering (Shanghai) Co., Ltd.
  • SPP1-REV 5'-aacaaaacatcacaccgtacc-3 '(SEQ ID NO. 87).
  • the cells to be edited are human adipose-derived mesenchymal stem cells (ADSC cells). According to the AAV tissue affinity control table, a packaging system of serotype AAV-9 is preferred.
  • the total packaging capacity of AAV-9 is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO. 66, in which the 433-435 bases are mutated PAM sites.
  • the sequence of the right homology arm is SEQ ID NO.67.
  • the inserts were T2A and EGFP, a total of 777bp, and the sequence is shown in SEQ ID NO.88.
  • Use seamless clone High-fidelity DNA assembly kit, purchased from NEB (Beijing) Co., Ltd., article number: E2621S). Cassette was inserted into the pAAV vector, and the vector sequence was SEQ ID NO.89.
  • PrimerStar high-fidelity DNA polymerase purchased from Bao Biological Engineering (Dalian) Co., Ltd., article number: R044A was used to engrav the loop.
  • the method introduced a point mutation (TGG to TCG) at the PAM site, thereby avoiding the cleavage of the recognition site by the SpCas9 / sgRNA RNP complex.
  • Point mutation primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • SPP1-PAM-mut-FW 5'-gtagaccccaaaagtaaagaagataaacacct-3 '(SEQ ID NO. 90).
  • SPP1-PAM-mut-REV 5'-aggtgtttatcttcttacttttggggtctac-3 '(SEQ ID NO. 91).
  • HEK293T cells were seeded in a quantity of 5 ⁇ 10 6 per dish to a 10 cm diameter containing 10 mL of complete medium (DMEM + 10% FBS + 1% P / S double antibody) (DMEM medium, CORNING petri dish purchased from ThermoFisher Scientific, Inc., article number: C11995500BT; FBS, purchased from ThermoFisher Scientific, Inc., article number: sv30087.02; P / S dual antibody, purchased from ThermoFisher Scientific, Inc., article number: SV30010) In total, 30 dishes were planted. The cells were cultured in a 37 ° C, 5% CO 2 cell incubator for 24 hours.
  • complete medium DMEM + 10% FBS + 1% P / S double antibody
  • the collected supernatant was centrifuged at 4000 rpm at 4 degrees for 10 minutes, and the impurities were discarded.
  • the impurity-removed supernatant was added to an Amicon Ultra-15 column (purchased from Merck Chemical Technology (Shanghai) Co., Ltd., article number: UFC 905096). After several centrifugations at 4000 rpm and 4 degrees for 30 minutes, the volume was concentrated to 10 to 15mL.
  • HEK293T cells scraped with a cell spatula were blown with an appropriate amount of medium and transferred to a 50 mL centrifuge tube. After centrifugation at 1500 rpm and 4 degrees for 10 min, the supernatant was discarded.
  • a total of 3 mL of cell lysis buffer (150 mM NaCl, 20 mM tris was added to all the pellets. (pH 8.0).
  • the resuspended cells were repeatedly freeze-thawed three times in a -80 ° C alcohol bath and a 37 ° C water bath.
  • the concentrated supernatant and the freeze-thawed cell suspension were mixed and 1 M MgCl 2 was added to a final concentration of 1 mM.
  • Add Benzonase (purchased from Merck Chemicals (Shanghai) Co., Ltd., article number: 70746-1kU) to a final concentration of 25U / mL, and mix for 40 minutes at 37 ° C after mixing. Take out the 50mL centrifuge tube, centrifuge at 4 °C, 4000rpm for 20min, and take the supernatant.
  • the virus was purified by iodixanol density gradient centrifugation.
  • AAV9 was tested for titer by qPCR.
  • the primers were designed based on the EGFP sequence so that the length of the qPCR product was about 200bp.
  • qPCR primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • AAVGFPF 5'-tcagcttcaggcaccaccac-3 '(SEQ ID NO. 92).
  • AAVGFPR 5'-tgaacttgtggccgtttacgtcg-3 '(SEQ ID NO. 93).
  • SpCas9 final concentration: 300 ug / ml
  • SPP1sgRNA final concentration: 175 ug / ml
  • a stem cell suspension is prepared, and the cell density is adjusted to 5 ⁇ 10 7 / mL.
  • the Lonza 4D nuclear transfection system was used to select the CB150 mode (the electric field intensity was 150 V / cm, the single pulse time was 10 ms, the time interval between two adjacent pulses was 20 s, and the total number of pulses was 5 times) for electrical transfer.
  • the cells were transferred to a 24-well plate containing DMEM / F12 complete medium and cultured in a 37 ° C, 5% CO 2 cell incubator.
  • the size of the clone is equivalent to a coin under a ten-fold objective lens 12 to 14 days after the electric transfer. Do not allow the clones to continue to grow or intersect.
  • Add 120 ⁇ L of DMEM / F12 complete medium to each well of a 96-well plate and label plate O. Observe through a microscope in a clean bench, adjust the P200 pipette to 45 ⁇ L, scrape the clones with a pipette tip with a filter element, collect the cells with a pipette, and transfer to a small well of a 96-well plate.
  • Example 4-2 is used to describe a fourth preferred embodiment of the present disclosure.
  • COL1A1 sgRNA 5'-uggggcaccaacguccaaggguuuugagacuaaauagcaaguuaaaauaaggcuaguccguuaucaaauaaguggcaccgagucggugcuuuuuuuuuuuu-3 '. (SEQ ID NO.68.
  • sequences at positions 1-20 are recognition motifs, and the rest are tracrRNA.
  • the sgRNA was synthesized by Integrated DNA Technologies. USA and added 2-O-Me and 3-phosphorothioate to the second and third positions of the three bases at the 5 'end and the 3' end of the sequence, respectively. Modification.
  • COL1A1-REV 5'-gcagtctgagaaccccagg-3 ', (SEQ ID NO. 95).
  • the cells to be edited are adipose-derived mesenchymal (ADSC) stem cell lines.
  • ADSC adipose-derived mesenchymal stem cell lines.
  • AAV tissue affinity control table a packaging system with a serotype of AAV9 is preferred.
  • the total packaging capacity of AAV9 is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO.69, in which the bases 436-438 are mutated PAM sites, and the sequence of the right homology arm is SEQ ID NO.70.
  • the inserts were T2A and EGFP, a total of 777bp, and the sequence is shown in SEQ ID NO.88.
  • Use seamless clone High-fidelity DNA assembly kit, purchased from NEB (Beijing) Co., Ltd., article number: E2621S).
  • Cassette was inserted into the pAAV vector, and the vector sequence was SEQ ID NO.89.
  • PrimerStar high-fidelity DNA polymerase purchased from Bao Biological Engineering (Dalian) Co., Ltd., article number: R044A was used to engrav the loop.
  • the method introduced a point mutation (TGG to TCG) at the PAM site, thereby avoiding the cleavage of the recognition site by the SpCas9 / sgRNA RNP complex.
  • Point mutation primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • COL1A1-PAM-mut-FW 5'-cccatcatcgatgtggcacccttggacgttggtgc-3 '(SEQ ID NO. 96).
  • COL1A1-PAM-mut-REV 5'-gcaccaacgtccaagggtgccacatcgatgatggg-3 '(SEQ ID NO. 97).
  • Example 4-1 The method of Example 4-1 was followed, except that at the 35th minute after the electric transfer, AAV9 was gently added dropwise according to a MOI of 1 ⁇ 10 5 , and the addition was completed within 50 minutes after the electric transfer.
  • Test samples Example 4-1, 4-2 and Comparative Example 4-1 After electroporation and AAV infection on the fourth day after trypsin digestion, fresh DMEM / F12 complete medium was added and the stem cell suspension was resuspended. liquid.
  • T2A-EGFP sequence inserted at the target locus expresses green fluorescent protein
  • use a flow cytometer to detect and count the number of cells that can emit green fluorescence and the total number of cells. Dividing the number of green fluorescent cells by the total number of cells gives you the total editing efficiency.
  • Example 4-1 The editing efficiency of Example 4-1 is 79.4%, the editing efficiency of Example 4-2 is 75.3%, and the editing efficiency of Comparative Example 1 is 9.5%.
  • Test samples monoclonal cell lines (plates A and B) cultured in 96-well plates
  • Detection method Design primers outside the homology arms.
  • the primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • Example 4-1 and Comparative Example 4-1 SPP1-FW and SPP1-REV were used.
  • Example 4-2 COL1A1-FW and COL1A1-REV were used.
  • Example 4-1 and Comparative Example 4-1 only 1144 bp bands were non-editing cells, only 1921 bp were double allele-editing cells, and both 1144 bp bands and 1921 bp were single allele-editing cells. Mark and count the proportions of these three types of editing types separately.
  • Example 4-2 only the bands of about 1084 bp are non-editing cells, only 1861 bp are bi-allele-editing cells, and both the band of about 1084 bp and 1861 bp are single-allele-editing cells.
  • Example 4-1 editing efficiency allele double editing 62.3%, allele single editing 18.1%
  • comparative example 4-1 editing efficiency allele double editing 0.5%
  • Example 4-2 Editing efficiency double allele editing 68.5%, allele single editing 15.5%; it can be seen that the time interval between electroporation and viral transfection has a greater impact on the efficiency of gene knock-in, and it is preferred that after the electroporation In 5-20 minutes, when the suspension of AAV virus particles to be transfected is added to the electrotransformed material to perform transfection for 8-24 hours, the efficiency of gene knock-in is the highest.
  • the apoptotic mesenchymal stem cells obtained with EGFP knock-in obtained in Example 4-1 were seeded on a 6-well plate and replaced with an osteogenic induction solution (10 mmol / L ⁇ -glyceryl phosphate (purchased from Sigma-Aldrich, Cat. : G9422), 1 ⁇ 10 -7 mol / L dexamethasone (purchased from Sigma-Aldrich, article number: D4902), 50 ⁇ g / mL ascorbic acid (purchased from Sigma-Aldrich, article number: PHR1008), and the total volume of each well is 2 mL.
  • the osteogenic induction fluid was changed every 3 days.
  • the treated cells were collected in sequence on the 0th, 9th, 12th, 15th, 18th, and 27th days of induction.
  • the collected cells were fixed and then subjected to immunocytochemical staining (
  • the primary antibody was Anti-Osteopontin, purchased from Abbot (Shanghai) Trading Co., Ltd., article number: ab69498, and the corresponding fluorescent secondary antibody was Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555. (Purchased from ThermoFisher Scientific, Inc., article number: A-21424).
  • the stained cells were observed with a green fluorescence channel and a red fluorescence channel using a laser confocal microscope, and finally the layers were merged (overlaid) and compared.
  • Table 4-2 shows.
  • the results in Table 4-2 above show that the model established in Example 4-1 can detect the induced bone differentiation in real time without causing cell death.
  • the expression of SPP1 gene is one of the signs of osteocyte differentiation and maturation.
  • the expression of the SPP1 gene is conventionally detected by immunochemical methods.
  • the results of real-time green fluorescence detection during the osteogenic differentiation of the EGFP knock-in adipose-derived stem cells constructed in Example 4-1 are consistent with the results of cellular immunochemical detection.
  • Example 4-2 The operation of Example 4-2 is the same as that of Example 4-1, and the corresponding primary antibody only needs to be replaced: Anti-Collagen I, purchased from Aibo (Shanghai) Trading Co., Ltd., article number: ab90395. The results are shown in Table 4-3.
  • Example 4-3 show that the model established in Example 4-2 can detect cell differentiation induced by real-time without causing cell death.
  • the expression of COL1A1 gene is one of the signs of osteocyte differentiation and maturation.
  • the expression of COL1A1 gene is routinely detected by immunochemical methods.
  • the EGFP knock-in adipose-derived mesenchymal stem cells constructed in Example 4-2 were real-time during the osteogenic differentiation process The results of green fluorescence detection are consistent with the results of cellular immunochemical detection.
  • Example 5-1 is used to describe the fifth preferred embodiment of the present disclosure in detail.
  • a cell model was constructed by inserting the EGFP reporter gene into the serum albumin gene (ALB) of placental mesenchymal stem cells. Using the ALB gene in situ promoter, insert P2A-EGFP before the stop codon.
  • ALB serum albumin gene
  • ALBsgRNA design tool http://crispr.mit.edu/ was used to design targeted recognition of ALB sgRNA on exon 14 of the ALB gene.
  • ALBsgRNA 5'-aaugugauguuauaagccuaguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuuuuuuuuu-3 (SEQ ID ID NO. 98).
  • the sequences at positions 1-20 are recognition motifs, and the rest are tracrRNA.
  • the sgRNA was synthesized by Integrated Technologies and USA, and modified by adding O-Me and phosphorothioate to the second and third positions of the three bases at the 5 'end and the 3' end of the sgRNA sequence, respectively. .
  • Genomic amplification recognizes the target sequence fragment (2000bp), and the primers are synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • ALB FW 5’-gagtctatttgtagaaaatg-3 ’(SEQ ID NO.104),
  • ALB REV 5'-ctctactgaagcgactggag-3 '(SEQ ID NO. 105).
  • the cells to be edited are human placental mesenchymal stem cells. According to the AAV tissue affinity control table, a packaging system of serotype AAV-9 is preferred.
  • the total packaging capacity of AAV-9 is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO. 99, in which the bases 490-492 are mutated PAM sites.
  • the sequence of the right homology arm is SEQ ID NO.100.
  • the inserts are P2A and EGFP, a total of 774bp, and the sequence is shown in SEQ ID NO.106.
  • Use seamless clone High-fidelity DNA assembly kit, purchased from NEB (Beijing) Co., Ltd., article number: E2621S). Cassette was inserted into the pAAV vector, and the sequence of the pAAV vector was SEQ ID NO.107.
  • HEK293T cells were seeded in a quantity of 5 ⁇ 10 6 per dish to a 10 cm diameter containing 10 mL of complete medium (DMEM + 10% FBS + 1% P / S double antibody) (DMEM medium, CORNING petri dish purchased from ThermoFisher Scientific, Inc., article number: C11995500BT; FBS, purchased from ThermoFisher Scientific, Inc., article number: sv30087.02; P / S dual antibody, purchased from ThermoFisher Scientific, Inc., article number: SV30010) In total, 30 dishes were planted. The cells were cultured in a 37 ° C, 5% CO 2 cell incubator for 24 hours.
  • complete medium DMEM + 10% FBS + 1% P / S double antibody
  • the collected supernatant was centrifuged at 4000 rpm at 4 degrees for 10 minutes, and the impurities were discarded.
  • the impurity-removed supernatant was added to an Amicon Ultra-15 column (purchased from Merck Chemical Technology (Shanghai) Co., Ltd., article number: UFC905096), and the volume was concentrated to 10 to 15 mL after several centrifugations at 4000 rpm and 4 degrees for 30 minutes. .
  • HEK293T cells scraped with a cell spatula were blown with an appropriate amount of medium and transferred to a 50 mL centrifuge tube. After centrifugation at 1500 rpm and 4 degrees for 10 min, the supernatant was discarded.
  • a total of 3 mL of cell lysis buffer (150 mM NaCl, 20 mM tris was added to all the pellets. (pH 8.0).
  • the resuspended cells were repeatedly freeze-thawed three times in a -80 ° C alcohol bath and a 37 ° C water bath.
  • the concentrated supernatant and the freeze-thawed cell suspension were mixed and 1 M MgCl 2 was added to a final concentration of 1 mM.
  • Add Benzonase (purchased from Merck Chemicals (Shanghai) Co., Ltd., article number: 70746-1kU) to a final concentration of 25U / mL, and mix for 40 minutes at 37 ° C after mixing. Take out the 50mL centrifuge tube, centrifuge at 4 °C, 4000rpm for 20min, and take the supernatant.
  • the virus was purified by iodixanol density gradient centrifugation.
  • AAV9 was tested for titer by qPCR.
  • the primers were designed based on the EGFP sequence so that the length of the qPCR product was about 200bp.
  • qPCR primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • AAVGFPF 5'-tcagcttcaggcaccaccac-3 '(SEQ ID NO. 108).
  • AAVGFPR 5'-tgaacttgtggccgtttacgtcg-3 '(SEQ ID NO.109).
  • SpCas9 final concentration: 300 ⁇ g / ml
  • ALB sgRNA final concentration: 175 ⁇ g / ml
  • the size of the clone is equivalent to a coin under a ten-fold objective lens 12 to 14 days after the electric transfer. Do not allow the clones to continue to grow or intersect.
  • Add 120 ⁇ L of DMEM / F12 complete medium to each well of a 96-well plate and label plate O. Observe through a microscope in a clean bench, adjust the P200 pipette to 45 ⁇ L, scrape the clones with a pipette tip with a filter element, collect the cells with a pipette, and transfer to the wells of a 96-well plate.
  • a cell model was constructed in which placental mesenchymal stem cells and a fetal globulin gene (AFP) were inserted with the EGFP reporter gene.
  • AFP gene in situ promoter was used, and P2A-EGFP was inserted before the stop codon. Since the two clip variants of the AFP have the same CTD, both clip variants of the AFP are marked at the same time in this embodiment. .
  • AFP sgRNA 5'-gguuguagaccccaaaaguaguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuuuu-3 '. (SEQ ID NO.101).
  • sequences at positions 1-20 are recognition motifs, and the rest are tracrRNA.
  • the sgRNA was synthesized by Integrated Technologies, USA and added to the second and third positions of the three bases at the 5 'end and the 3' end of the sequence by adding 2-O-Me and -3-phosphorothioate, respectively. Modification.
  • the cells to be edited are placental mesenchymal stem cell lines. According to the AAV tissue affinity control table, a packaging system with a serotype of AAV9 is preferred.
  • the total packaging capacity of AAV9 is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO. 102, of which the 493-495 bases are the mutated pam site), and the sequence of the right homology arm is SEQ ID NO. 103.
  • the inserts were P2A and EGFP, a total of 774bp, and the sequence is shown in SEQ ID NO.108.
  • the method introduced a point mutation (CGG to CTG) at the PAM site, thereby avoiding the cleavage of the recognition site by the SpCas9 / sgRNA RNP complex.
  • Point mutation primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • AFP-PAM-mut-FW 5'-aaactcgtgctgctttaggagtttaaattactt-3 '(SEQ ID NO. 112).
  • AFP-PAM-mut-Rev 5'-aagtaatttaaactcctaaagcagcacgagttt-3 '(SEQ ID NO. 113).
  • Example 5-1 The method of Example 5-1 was performed, except that at the 35th minute after the electric transfer, AAV9 was gently added dropwise according to a MOI of 1 ⁇ 10 5 and the addition was completed within 50 minutes after the electric transfer.
  • Test samples Examples 5-1, 5-2 and Comparative Example 5-1 After electroporation and AAV infection on the fourth day after trypsin digestion, fresh DMEM / F12 complete medium was added and the stem cells were resuspended. liquid.
  • Detection method Because the P2A-EGFP sequence inserted at the target locus expresses green fluorescent protein, use a flow cytometer to detect and count the number of cells that can emit green fluorescence and the total number of cells. Dividing the number of cells that can emit green fluorescence by the total number of cells gives you the total editing efficiency.
  • Example 5-1 The editing efficiency of Example 5-1 is 78.1%, the editing efficiency of Example 5-2 is 76.6%, and the editing efficiency of Comparative Example 5-1 is 1.2%.
  • Test samples monoclonal cell lines (plates A and B) cultured in 96-well plates
  • Detection method Design primers outside the homology arms.
  • the primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • Example 5-1 and Comparative Example 5-1 ALB-FW and ALB-REV were used.
  • Example 5-2 AFP-FW and AFP-REV were used.
  • Example 5-1 Comparative Example 5-1 Only those with a band of about 2000 bp were non-editing cells, only 2800 bp were bi-allele-editing cells, and both the band of about 2000 bp and 2800 bp were single-allele-editing cells. Mark and count the proportions of these three types of editing types separately.
  • Example 5-2 only bands of about 2000 bp were non-editing cells, only 2800 bp were bi-allele-editing cells, and both bands of about 2000 bp and 2800 bp were mono-allele-editing cells. Mark and count the proportions of these three types of editing types separately.
  • Example 5-1 editing efficiency double allele editing 30.2%, allele single editing 55.3%, comparative example 5-1 editing efficiency: allele double editing 0.9%, allele single editing 1.5%
  • Example 5-1 placental mesenchymal stem cells with EGFP in ALB and Example 2 in AFP.
  • EGFP is induced on placental mesenchymal stem cells to differentiate into mature liver-like cells.
  • the fourth induction medium to induce cell differentiation and maturation (total induction days 13, 15, 17, 19, and 21)
  • the cells were fixed and then immunocytochemical stained (the primary antibody of ALB was purchased as an Anti-Albumin antibody).
  • the corresponding fluorescent secondary antibody is Goat anti-Chicken, IgY (H + L) Secondary Antibody, Alexa Fluor, purchased from ThermoFisher Scientific, Inc., article number: A-21424;
  • the primary antibody of AFP is: Anti-alpha 1Fetoprotein antibody [AFP-01] was purchased from Abbot (Shanghai) Trading Co., Ltd., article number: ab3980, and the corresponding fluorescent secondary antibody was Goat anti-Mouse IgG (H + L) Highly Cross- Adsorbed Secondary Antibody, Alexa Fluor Plus Plus 555 was purchased from Thermo Fisher Scientific, Inc., article number: A-21424). Observe the green fluorescence channel and the red fluorescence channel of the two groups of cells using a fluorescence microscope, and then merge the layers to measure the coincidence rate of the green fluorescence and red fluorescence observations.
  • the cells indicated by EGFP are consistent with the cells indicated by ALB in Example 5-1 and AFP indicated in Example 5-2. That is to say, in the EGFP-labeled placental mesenchymal stem cells established in Example 5-1 and Example 5-2, the degree of liver-like differentiation can be detected in real time. The degree of liver-like differentiation can be detected in real time. Degree of liver-like differentiation.
  • Example 6-1 is used to describe a sixth preferred embodiment of the present disclosure.
  • RNA45SN4 a GPX7 gene-expressing Cassette was inserted into a safe site (RNA45SN4) of a 1016 cell line (purchased from Harvard University Stem Cell Bank) was constructed.
  • RNA45SN4sgRNA designed by using the online sgRNA design tool (http://crispr.mit.edu/). The preferred results are as follows: .114). The sequences at positions 1-20 are recognition motifs, and the rest are tracrRNA.
  • the sgRNA was synthesized by Integrated DNA Technologies and US company and added O-Me and phosphorothioate to the second and third positions of the three bases at the 5 'end and the 3' end of the sgRNA sequence. .
  • the cell to be edited is a 1016 cell line.
  • a packaging system with a serotype of AAV-8 is preferred.
  • the total packaging capacity of AAV-8 is 4.7Kb.
  • the fragment inserted on the loading vector of the AAV packaging system should include the left homology arm (about 500 bp), the insertion fragment, and the right homology arm (about 500 bp).
  • the sequence of the left homology arm is SEQ ID NO.115.
  • the sequence of the right homology arm is SEQ ID NO. 116, in which bases 4-7 are mutated PAM sites.
  • the inserted fragment is a complete Cassette, including CMV increaser, CMV promoter, GPX7CDS and bGH poly A, a total of 1296bp, the sequence is shown in SEQ ID NO.4, where the 1-507th is the promoter, the 508-1071th EGFP, bGH polyA at positions 1072-1296.
  • Cassette was inserted into the pAAV vector using NheI and BsmI restriction enzymes (purchased from NEB (Beijing) Co., Ltd.), and the vector sequence is SEQ ID NO.7.
  • PrimerStar high-fidelity DNA polymerase purchased from Bao Biotechnology (Dalian) Co., Ltd., article number: R044A was used to engrav the loop.
  • the method introduced a point mutation (TGG to TAG) at the PAM site, thereby avoiding the cleavage of the recognition site by the SpCas9 / sgRNA and RNP complex.
  • Point mutation primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • GPx7-PAM-mut-FW 5’-gcggcggccgtcgggtaggggctttacccggcg-3 ’(SEQ ID NO. 121);
  • GPx7-PAM-mut-REV 5'-cgccgggtaaagcccctacccgacggccgccgc-3 '(SEQ ID NO. 122).
  • HEK293T cells were seeded in a quantity of 5 ⁇ 10 6 per dish to a 10 cm diameter containing 10 mL of complete medium (DMEM + 10% FBS + 1% P / S double antibody) (DMEM medium, CORNING petri dish purchased from ThermoFisher Scientific, Inc., article number: C11995500BT; FBS, purchased from ThermoFisher Scientific, Inc., article number: sv30087.02; P / S dual antibody, purchased from ThermoFisher Scientific, Inc., article number: SV30010) In total, 30 dishes were planted. The cells were cultured in a 37 ° C, 5% CO 2 cell incubator for 24 hours.
  • complete medium DMEM + 10% FBS + 1% P / S double antibody
  • the collected supernatant was centrifuged at 4000 rpm at 4 degrees for 10 minutes, and the impurities were discarded.
  • the impurity-removed supernatant was added to an Amicon Ultra-15 column (purchased from Merck Chemical Technology (Shanghai) Co., Ltd., article number: UFC 905096). After several centrifugations at 4000 rpm and 4 degrees for 30 minutes, the volume was concentrated to 10 to 15mL.
  • HEK293T cells scraped with a cell spatula were blown with an appropriate amount of medium and transferred to a 50 mL centrifuge tube. After centrifugation at 1500 rpm and 4 degrees for 10 min, the supernatant was discarded.
  • a total of 3 mL of cell lysis buffer (150 mM NaCl, 20 mM tris was added to all the pellets. (pH 8.0).
  • the resuspended cells were repeatedly freeze-thawed three times in a -80 ° C alcohol bath and a 37 ° C water bath.
  • the concentrated supernatant and the freeze-thawed cell suspension were mixed and 1 M MgCl 2 was added to a final concentration of 1 mM.
  • Add Benzonase (purchased from Merck Chemicals (Shanghai) Co., Ltd., article number: 70746-1kU) to a final concentration of 25U / mL, and mix for 40 minutes at 37 ° C after mixing. Take out the 50mL centrifuge tube, centrifuge at 4 °C, 4000rpm for 20min, and take the supernatant.
  • the virus was purified by iodixanol density gradient centrifugation.
  • AAV8 was tested for titer by qPCR.
  • the primers were designed based on the EGFP sequence so that the length of the qPCR product was about 200bp.
  • qPCR primers were synthesized by Biotech Bioengineering (Shanghai) Co., Ltd.
  • AAV-GPx7-F 5'-actggtgtcgctggagaagt-3 '(SEQ ID NO. 123).
  • AAV-GPx7-R 5'-caatctccttgttgctgtcag-3 '(SEQ ID NO. 124).
  • SpCas9 final concentration 1 ⁇ mol / ⁇ L
  • RNA45SN4sgRNA final concentration 1 ⁇ mol / ⁇ L
  • cells were transferred to geltrex-coated (purchased from ThermoFisher Scientific, Inc., article number: A1413202) and added with 500 ⁇ L of mTeSR and 10 ⁇ M Y-27632 (purchased from Stem Cell Technologies, Inc., item: 72304). The culture was continued in a 24-well plate in a 37 ° C, 5% CO 2 cell incubator.
  • AAV-DJ is gently added at a MOI value of 1 ⁇ 10 5 within 5 to 20 minutes after the electric transfer, and the addition is completed within 20 minutes after the electric transfer.
  • Cells were seeded at a rate of 15000 cells per 10cm dish into a 10cm CORNING dish coated with Geltrex and added with 10mL mTeSR and 10 ⁇ M Y-27632 in a 37 ° C, 5% CO 2 cell culture incubator. .
  • the size of the clone is equivalent to a coin under a ten-fold objective lens 12 to 14 days after the electric transfer. Do not allow the clones to continue to grow or intersect.
  • 120 ⁇ L mTeSR (containing 10 ⁇ M Y-27632) was added to each well of a 96-well plate coated with Geltrex, and plate O was labeled. Observe through a microscope in a clean bench, adjust the P200 pipette to 45 ⁇ L, scrape the clones with a pipette tip with a filter element, collect the cells with a pipette, and transfer to the wells of a 96-well plate.
  • Pluripotent stem cells are induced to differentiate into MSCs
  • the obtained embryoid bodies were seeded in a matrigel (geltrex, purchased from Thermo Fisher Scientific, Inc.) coated six-well plate for culture, and the culture was continued for 2 weeks until the appearance of fibrous cells. After another passaging, flow cytometry was used to sort the cell populations in which CD73, CD90, and CD105 were all positive. This is the GPx7 enhanced MSC.
  • Cell senescence ⁇ -galactosidase staining is a method for staining senescent cells or tissues based on the upregulation of SA- ⁇ -Gal activity level during aging.
  • SA- ⁇ -Gal staining was performed on the GPx7 enhanced MSC obtained in Example 6-1 and the umbilical cord mesenchymal stem cells of Comparative Example 6-1, respectively.
  • the aging of cells or tissues was observed under a light microscope, and the staining ratio of SA- ⁇ -Gal positive cells in the two groups of cells was quantitatively analyzed.
  • Staining step Wash the donor cells once with PBS, then add staining fixative (2% formaldehyde + 0.2% glutaraldehyde), and fix for 5 minutes at room temperature. Discard the fixative solution, wash it once with PBS, and add 1 ml of staining working solution to each well. Using X-Gal as a substrate, beta-galactosidase, which is specific for aging, produces a dark blue product. Experimental results: Positive ratio of senescent cells: Comparative Example 6-1> Example 6-1. The results are shown in Table 6-2.
  • the GPx7-enhanced MSC obtained in Example 6-1 and the umbilical cord mesenchymal stem cells of Comparative Example 6-1 were passaged for 10 to 15 passages in vitro to perform protein expression on the senescence-highly expressed genes p16, p21, and GATA4, respectively.
  • the primary antibodies are p16 (purchased from BD, article number: 550834), p21 (purchased from Cell Signaling, article number: 2947), GATA4 (purchased from Santa Cruz, article number: SC-1237), Actin (purchased from Sigma-Aldrich, article number : A1978).
  • the HRP-labeled secondary antibodies used were purchased from Nakasugi Jinqiao as goat anti-mouse, article number: ZDR5307, goat anti-rabbit, article number: ZDR5306, rabbit anti-sheep, article number: ZDR5308.
  • STZ rats were modeled according to the method described in "Effects and mechanisms of adipose-derived mesenchymal stem cells and their exosomes on inflammatory adipocytes and type 2 diabetic rats".
  • the GPx7-enhanced MSC cells obtained in Example 1 and the umbilical cord mesenchymal stem cells of Comparative Example 1 were injected into STZ rats through the tail vein at a concentration of 1 ml at a dose of 2 x 10 6 cells / ml, and the cells were injected for 5 days. Blood was taken from the tail vein at 10 days, 15 days, and 20 days, and fasting blood glucose concentrations were measured. The results are shown in Table 6-4
  • Example 6-1 As shown in Table 6-4, compared with Comparative Example 6-1, Example 6-1 has the ability to maintain the blood glucose regulating effect for a long time.

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Abstract

提供了一种在干细胞中定向敲入基因的方法，包括如下步骤：S1、将针对待敲入的靶位点的经化学修饰的sgRNA和Cas9蛋白混合以形成RNP复合物；S2、形成待转染AAV病毒颗粒；S3、将干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；S4、向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行的转染，得到转染后的培养物；S5、将所述转染后的培养物极限稀释后进行单克隆化培养，通过PCR及测序并筛选出进行了基因定向敲入的单克隆细胞株。该方法无需筛选标记，可显著提高大片段基因敲入干细胞的效率。

Description

一种在干细胞中进行基因定向敲入的方法 技术领域
本公开涉及生物技术领域，具体地，涉及一种在干细胞中进行基因定向敲入的方法。
背景技术
CRISPR基因编辑技术通过在基因组特定位点进行精确的靶向性剪切，形成DNA双链缺口(Double-strand breaks，DSBs)。DSBs可激活细胞内非同源末端链接(Non-homologous end joining，NHEJ)和同源重组(Homology directed repair，HDR)两种修复机制对DNA进行修复。在哺乳动物细胞中，NHEJ修复基因占主导模式，可引入碱基的随机插入或缺失，实现基因敲除。HDR则需要在外源基因模板存在的情况下，实现对基因组的精确修复。
人多能干细胞因其具有多能性可成为体外不同类型的成体细胞和器官小体的潜在来源，其快速增殖能力使其可为科研分析及大规模药物筛选提供充足原材料。基因编辑技术在人多能干细胞中的应用对实现疾病模拟和推动再生医学的发展具有重要意义，已成为再生医学领域研究的热点。但在多能干细胞中，通过Cas9蛋白剪切实现HDR精准编辑基因组的效率极低，在多能干细胞中提高基因敲入效率成为亟待解决的关键技术问题。2014年Gonzalez研究组报道发现，通过在Cas9人多能干细胞中同时转入gRNA和ssDNA模板，可实现在人多能干细胞单个碱基基因敲入的效率达到10％。同年，另一研究组报道，在用诺考达唑(nocodazale)药物处理细胞使细胞处于M期之后，将Cas9核糖蛋白与ssDNA转染至人胚胎干细胞(hESCs)，基因敲入效率可达1.6％。2017年有研究发现，在CRISPR/Cas9体系下，使用单链脱氧寡核苷酸(ssODNs)作为模板，对10bp以下基因片段进行敲入人iPSC细胞株的效率可达45％。但由于ssODNs的大小与基因编辑效率存在负相关，Cas9/ssODN技术难以实现基因片段长度超过90bp的基因敲入。2017年发明的iCRISPR体系，在hESCs中对小片段基因敲入效率可达30％，但应用于大片段敲入效率则显著下降，只有1％不到的HDR整合率。
另外，由于目前在多能干细胞中基因编辑效率低，通常需要通过筛选标记进行筛选来获得单克隆阳性细胞株，而筛选标记有可能会影响细胞功能，限制其在研究及临床上的应用。虽然通过piggyBac或Cre/loxP体系可以删除筛选标记，但Cre/loxP体系会将大片段的loxP序列滞留在基因组上，piggyBac体系则需要附近存在TTAA位点来防止其他外源序列的引入。
发明内容
本公开的目的是实现一种可显著提高大片段基因敲入干细胞效率的技术方案。
为了实现上述目的，本公开提供了一种在干细胞中进行基因定向敲入的方法，该方法包括如下步骤：S1、将针对待敲入的靶位点的sgRNA和Cas9蛋白混合以形成RNP复合物；S2、将插入有模板DNA的同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；S3、将干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
通过上述技术方案，本发明建立了一种可显著提高大片段基因敲入人干细胞效率的技术方案，并可在敲入报告基因的情况下进一步建立无需筛选标记的可用于检测不同干细胞株敲入大片段基因效率的方法，通过此方法可检测不同干细胞株中不同基因位点的基因敲入效率，从而选择最佳的科研及临床应用方案。
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。
具体实施方式
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。
本公开提供了一种在干细胞中进行基因定向敲入的方法，该方法包括如下步骤：S1、将针对待敲入的靶位点的sgRNA和Cas9蛋白混合以形成RNP复合物；S2、将插入有模板DNA的同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；S3、将干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
其中，电转是指DNA的电转化，又称作高压电穿孔法(high-voltage electroproration，简称电穿孔法electroproration)，可用于将DNA导入细胞。
本发明通过RNP复合物以电转的方式将sgRNA和Cas9蛋白转入细胞中，并在电转后的合适的时间内选择AAV病毒来将插入有模板DNA的载体转染入细胞中，最终使得sgRNA、Cas9蛋白和插入有模板DNA的载体共同通过CRISPR基因编辑来使得大片段的外源基因得以定向敲入靶点位置。
其中，可选地，所述sgRNA带有化学修饰基团；针对待编辑的靶位点的sgRNA和Cas9蛋白的用量摩尔比为1:1至1:5。其中，可以使用在线sgRNA设计工具(http://crispr.mit.edu/)依据待编辑的靶位点设计sgRNA的序列并加以合成。其中，sgRNA序列的5’端和3’端末尾的可以分别添加有甲基(-O-Me)化学修饰基团或磷硫酰(-phosphorothioate)化学修饰基团。
其中，可选地，将针对待编辑的靶位点的sgRNA和Cas9蛋白混合的时间为5-20分钟，温度为10-40℃。
其中，可选地，步骤S3中，所述干细胞的悬液与所述RNP复合物混合时，相对于每10 6个所述干细胞，以sgRNA的量计，所述RNP复合物的用量为1-50μmol；所述干细胞的悬液中，细胞浓度为(1-5)×10 7个/mL；以sgRNA的量计，所述RNP复合物的终浓度为0.1-1.5μmol/μL。
其中，可选地，步骤S3中，电转的条件包括：电场强度为50-250V/cm，单次脉冲时间为2-15ms，相邻两次脉冲之间的时间间隔为10-60s，总脉冲次数为2-10次。
其中，可选地，步骤S4中，在电转结束后5-20分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行8-24小时的转染，得到转染后的培养物。
其中，可选地，步骤S4中，待转染的AAV病毒颗粒的悬液的加入量使得待转染的AAV病毒颗粒的MOI值为10 4-10 6。MOI值是感染时病毒与细胞数量的比值。
其中，优选地，所述干细胞可以为胚胎干细胞、诱导多能干细胞和间充质干细胞中的至少一种；优选所述干细胞为人诱导多能干细胞1016细胞株。所述AAV病毒的血清型为AAV-6病毒、AAV-1病毒或AAV-DJ病毒，优选所述AAV病毒的血清型为AAV-DJ病毒，在该优选情况下，能够进一步增加大片段基因敲入干细胞的效率。
其中，所述模板DNA可以包括左同源臂序列、敲入片段和右同源臂序列；所述左同源臂序列和所述右同源臂序列共同决定了插入的精确位点，可以通过所述左同源臂序列和所述右同源臂序列来精确控制敲入片段插入的位点就是待敲入的靶位点。
优选地，所述敲入片段包括增强子、启动子、敲入基因和polyA。
根据本公开，所述敲入基因的长度可以为200-5000bp；相对于已有的定向敲入方法，在成功率相同的情况下，本公开的方法显著地提高了所述敲入基因的长度。
根据本公开提供的第一种优选实施方式，所述敲入基因为报告基因；在该优选情况下，可以无需筛选标记来检测不同干细胞株敲入大片段基因效率；进一步优选所述报告基因为荧光蛋白报告基因；所述荧光蛋白报告基因可以为EGFP报告基因。
根据本公开提供的第一种优选实施方式，可选地，所述待敲入的靶位点的sgRNA的序列如SEQ ID NO.1所示；所述模板DNA的序列如SEQ ID NO.6所示；所述载体的骨架序列如SEQ ID NO.7所示。所述载体的骨架序列是指未插入模板DNA的载体的序列。
为了实现对间充质干细胞衰老的实时检测，提供可实时检测间充质干细胞衰老的细胞模型以进行药物筛选和毒性检测，本公开提供了第二种优选实施方式。
根据本公开提供的第二种优选实施方式，所述在干细胞中进行基因定向敲入的方法为一种制备可实时检测间充质干细胞衰老的细胞模型的方法，该方法包括如下步骤：S1、将针对衰老相关基因的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；所述衰老相关基因包括Klotho、p16、p21、p53、GATA4、SIRT1、SIRT3、SIRT6和MORF4中的至少一种；所述衰老相关基因的敲入位点为所述衰老相关基因的最末一个有义密码子和终止密码子之间；S2、将插入有模板DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模板DNA包括针对所述衰老相关基因的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有自剪切2A肽编码序列和荧光蛋白报告基因，以使得定向敲入后形成编码由衰老相关基因、2A肽和荧光蛋白串联而成的融合蛋白的融合基因；S3、将间充质干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
根据本公开提供的第二种优选实施方式，本公开建立了一种可在间充质干细胞高效敲入长片段报告基因的方法，在不影响衰老相关基因表达的情况下，原位插入报告基因，由此可以构造多种与间充质干细胞衰老相关的细胞模型，并且进而可以实现在干细胞药物制备过程中，实时监测连续传代的细胞是否存在老化现象，并可在动物模型中检测制备的间充质干细胞药物移植至体内不同组织，处于疾病或衰老的组织微环境中，组织特异性的细胞因子及有害代谢产物是否会影响间充质干细胞在体内的衰老而影响细胞体内存活时间与功能，便于对间充质干细胞体内作用时间与有效性进行深入研究，促进其临床转化及工业化生产制备中的质量监测。
根据本公开提供的第二种优选实施方式，所述衰老基因是以NCBI数据库中的Gene Symbol来定义的，具体信息如表2-1所示，其对应的代表性sgRNA序列和左右同源臂序列也列于表2-1中。优选地，所述sgRNA如SEQ ID NO.14、17、20、23、26、29、32、35或38所示；相应地，所述左同源臂序列如SEQ ID NO.15、18、21、24、27、30、33、36或39所示；相应地，所述右同源臂序列如SEQ ID NO.16、19、22、25、28、31、34、37或40所示。
表2-1
Figure PCTCN2018119852-appb-000001
Figure PCTCN2018119852-appb-000002
通过本公开提供的第二种优选实施方式，优选地，所述间充质干细胞为骨髓间充质干细胞、脂肪间充质干细胞、脐带间充质干细胞、宫血间充质干细胞及牙髓间充质干细胞。所述AAV病毒的血清型为AAV-6病毒、AAV-1病毒或AAV9病毒，优选所述AAV病毒的血清型为AAV9病毒，在该优选情况下，能够进一步增加大片段基因敲入干细胞的效率。
根据本公开提供的第二种优选实施方式，其中，所述自剪切2A肽用于将衰老相关基因的蛋白和荧光蛋白切割开，其可以为T2A肽、F2A肽和P2A肽中的至少一种；所述荧光蛋白报告基因的选择可以较宽，例如可以为EGFP、ECFP、EYFP、ERFP、mCherry、tdTomato、Venus中的至少一种。
根据本公开提供的第二种优选实施方式，其中，可选地，所述荧光蛋白报告基因的序列如SEQ ID NO.43所示；所述载体的骨架序列如SEQ ID NO.44所示。所述载体的骨架序列是指未插入模板DNA的载体的序列。
为了克服间充质干细胞存在免疫抑制且对肿瘤的杀伤效率较低的缺陷，解除间充质干细胞的免疫抑制并提高间充质干细胞对肿瘤的杀伤效率，本公开提供了第三种优选实施方式。
根据本公开的第三种优选实施方式，在干细胞中进行基因定向敲入的方法为一种可解除免疫抑制并增强肿瘤靶向性杀伤的间充质干细胞的制备方法，该方法包括如下步骤：S1、将针对PD-1基因的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；S2、将插入有模版DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模版DNA包括针对PD-1基因的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有TRAIL基因；且能够通过TRAIL基因的定向敲入使得PD-1基因的表达沉默；S3、将间充质干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
通过本公开的第三种优选实施方式，本发明建立了一种可在间充质干细胞高效敲入长片段报告基因的方法，在间充质干细胞中敲除PD-1基因的同时插入肿瘤坏死因子相关凋亡诱导配体(TRAIL,TNF-related apoptosis-inducing ligand)基因，通过敲除PD-1实现对CD8 +T细胞抑制的解除(G0/G1期细胞数量减少，S期细胞数量增加)，通过敲入TRAIL实现选择性地诱导肿瘤细胞凋亡，制备了一种可解除免疫抑制并增强肿瘤靶向性杀伤的人间充质干细胞。本发明同时解决了肿瘤治疗中常见的微环境中存在免疫抑制及常规药物毒性大、不具有靶向性的问题。
根据本公开的第三种优选实施方式，其中，PD-1基因是以NCBI数据库中的Gene Symbol来定义的，其NCBI Gene ID为5133。
根据本公开的第三种优选实施方式，其中，所述PD-1基因的敲入位点能够使得通过TRAIL基因的定向敲入使得PD-1基因的表达沉默，优选所述PD-1基因的敲入位点位于PD-1基因的第一外显子上。
根据本公开的第三种优选实施方式，其中，所述TRAIL基因是指肿瘤坏死因子相关凋亡诱导配体(TRAIL，TNF-related apoptosis-inducing ligand)基因，可以为膜结合型TRAIL基因或分泌型TRAIL基因。
根据本公开的第三种优选实施方式，其中，所述模版DNA可以包括左同源臂序列、敲入片段和右同源臂序列；优选地，所述左同源臂序列和所述右同源臂序列之间可以插入有CMV增强子、 CMV启动子、TRAIL基因的表达框和SV40poly A；优选地，所述左同源臂序列和所述右同源臂序列之间插入的序列如SEQ ID NO.56或SEQ ID NO.62所示。
根据本公开的第三种优选实施方式，其中，优选地，所述sgRNA如SEQ ID NO.53所示；相应地，所述左同源臂序列如SEQ ID NO.54所示；相应地，所述右同源臂序列如SEQ ID NO.55所示。
根据本公开的第三种优选实施方式，其中，可选地，所述载体的骨架序列如SEQ ID NO.59所示。所述载体的骨架序列是指未插入模版DNA的载体的序列。
为了实现对间充质干细胞骨分化的实时检测，提供可实时检测间充质干细胞骨分化的细胞模型以进行药物筛选，本公开提供了第四种优选实施方式。
根据本公开的第四种优选实施方式，在干细胞中进行基因定向敲入的方法为一种制备可实时检测间充质干细胞骨分化的细胞模型的方法，该方法包括如下步骤：S1、将针对骨分化相关基因的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；所述骨分化相关基因包括SPP1、COL1A1、BMP-2、Runx2、SLP1、IBSP及BGLAP中的至少一种；所述骨分化相关基因的敲入位点为所述骨分化相关基因的最末一个有义密码子和终止密码子之间；S2、将插入有模板DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模板DNA包括针对所述骨分化相关基因的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有自剪切2A肽编码序列和荧光蛋白报告基因，以使得定向敲入后形成编码由骨分化相关基因、2A肽和荧光蛋白串联而成的融合蛋白的融合基因；S3、将间充质干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
通过本公开的第四种具体实施方式，建立了一种可在间充质干细胞高效敲入长片段报告基因的方法，在不影响骨分化相关基因表达的情况下，原位插入报告基因，由此可以构造多种与间充质干细胞骨分化相关的细胞模型，并且进而可以实现在干细胞药物制备过程中，实时监测连续传代的细胞成骨分化状态，并可在动物模型中检测制备的间充质干细胞药物移植至体内不同组织，处于疾病或骨分化的组织微环境中，组织特异性的细胞因子及有害代谢产物是否会影响间充质干细胞在体内的骨分化而影响细胞体内存活时间与功能，便于对间充质干细胞体内作用时间与有效性进行深入研究，促进其临床转化及工业化生产制备中的质量监测。
根据本公开的第四种具体实施方式，其中，所述骨分化基因是以NCBI数据库中的Gene Symbol来定义的，具体信息如表4-1所示，其对应的代表性sgRNA序列和左右同源臂序列也列于表4-1中。优选地，所述sgRNA如SEQ ID NO.65、68、71、74、77、80或83所示；相应地，所述左同源臂序列如SEQ ID NO.66、69、72、75、78、81或84所示；相应地，所述右同源臂序列如SEQ ID NO.67、70、73、76、79、82或85所示。
表4-1
Figure PCTCN2018119852-appb-000003
根据本公开的第四种具体实施方式，其中，所述自剪切2A肽用于将骨分化相关基因的蛋白和荧光蛋白切割开，其可以为T2A肽、F2A肽和P2A肽中的至少一种；所述荧光蛋白报告基因的选择可以较宽，例如可以为EGFP、ECFP、EYFP、GFP、RFP、mCherry、tdTomato和Venus中的至少一种。
根据本公开的第四种具体实施方式，其中，可选地，所述荧光蛋白报告基因的序列如SEQ ID NO.88所示；所述载体的骨架序列如SEQ ID NO.89所示。所述载体的骨架序列是指未插入模板DNA的载体的序列。
为了实现对间充质干细胞向成熟肝样细胞分化的实时检测，提供可实时检测间充质干细胞向成熟肝样细胞分化的细胞模型以进行药物筛选，本公开提供了第五种优选的实施方式。
根据本公开提供的第五种优选的实施方式，在干细胞中进行基因定向敲入的方法为一种制备可实时检测间充质干细胞向成熟肝样细胞分化的细胞模型的方法，该方法包括如下步骤：S1、将针对肝样细胞相关基因的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；所述肝样细胞相关基因包括ALB和/或AFP；所述肝样细胞相关基因的敲入位点为所述肝样细胞相关基因的最末一个有义密码子和终止密码子之间；S2、将插入有模板DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模板DNA包括针对所述肝样细胞相关基因的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有自剪切2A肽编码序列和荧光蛋白报告基因，以使得定向敲入后形成编码由肝样细胞相关基因、2A肽和荧光蛋白串联而成的融合蛋白的融合基因；S3、将间充质干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
通过本公开提供的第五种优选的实施方式，本公开建立了一种可在间充质干细胞高效敲入长片段报告基因的方法，在不影响肝脏分化成熟标志物ALB基因和肝脏未成熟标准物AFP的基因表达的情况下，原位插入报告基因，由此可以构造了可在干细胞药物制备过程中，实时监测诱导分化的间充质干细胞向成熟肝样细胞分化的分化成熟情况的细胞模型，可简便、快速、直观的检测间充质干细胞诱导向成熟肝样细胞分化的分化成熟情况，便于对间充质干细胞作为药物制备的整体过程进行质控、分析及质量判定，推动建立其工业化制备的标准，促进其临床转化。
根据本公开提供的第五种优选的实施方式，其中，所述肝样细胞相关基因是以NCBI数据库中的Gene Symbol来定义的，具体信息如表5-1所示，其对应的代表性sgRNA序列和左右同源臂序列也列于表5-1中。优选地，所述sgRNA如SEQ ID NO.98或101所示；相应地，所述左同源臂序列如SEQ ID NO.99或102所示；相应地，所述右同源臂序列如SEQ ID NO.100或103所示。
表5-1
Figure PCTCN2018119852-appb-000004
其中，所述自剪切2A肽用于将肝样细胞相关基因的蛋白和荧光蛋白切割开，其可以为T2A肽、F2A肽和P2A肽中的至少一种；所述荧光蛋白报告基因的选择可以较宽，例如可以为EGFP、ECFP、EYFP、GFP、RFP、mCherry、tdTomato和Venus中的至少一种。
其中，可选地，所述荧光蛋白报告基因的序列如SEQ ID NO.106所示；所述载体的骨架序列 如SEQ ID NO.10107所示。所述载体的骨架序列是指未插入模板DNA的载体的序列。
为了克服间充质干细胞存在的易衰老的缺陷，获得一种能够抵抗细胞衰老及延长血糖调节作用时程的细胞治疗产品，本公开提供了第六种优选的实施方式。
根据本公开的第六种优选的实施方式，本公开提供了一种抵抗细胞衰老及延长血糖调节作用时程的间充质干细胞的制备方法，该方法包括如下步骤：S1、将针对基因插入安全岛的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；S2、将插入有模板DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模板DNA包括针对基因插入安全岛的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有GPx7基因；S3、将iPSC细胞或胚胎干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株；S6、将所述进行了基因定向敲入的单克隆细胞株扩增并定向诱导分化成为间充质干细胞。
基于本公开的第六种优选的实施方式，本公开还提供了上述方法制备得到的间充质干细胞。
基于本公开的第六种优选的实施方式，本公开还提供了上述间充质干细胞的用途，所述用途为如下任意一种：制备改善胰岛素抵抗和/或慢性炎症的产品；制备细胞移植治疗的产品；制备改善β细胞功能的治疗性产品；制备再生和/或修复损伤血管的产品。
通过本公开的第六种优选的实施方式，本公开在间充质干细胞的基因插入安全岛(包括rDNA28s、rDNA 18S、rDNA45s、CLYBL及AAVS1)部位精确、高效插入GPx7表达元件，来提升GPx7表达量从而延缓间充质干细胞的复制性衰老，从而延长间充质干细胞的调节血糖的时间。
根据本公开的第六种优选的实施方式，其中，所述基因插入安全岛的定义为外源基因安全插入位点，外源基因插入后并不会影响细胞本身的基因表达；所述基因插入安全岛可以为RNA45SN4、rDNA 28S(NCBI Gene ID为106632264)、rDNA 18S(NCBI Gene ID为106631781)、rDNA 45S(NCBI Gene ID为109864271)、CLYBL(NCBI Gene ID为171425)或AAVS1(NCBI Gene ID为17)；所述插入基因GPx7，其NCBI Gene ID为2882。优选地，所述基因插入安全岛为RNA45SN4，其NCBI Gene ID为109864271。
根据本公开的第六种优选的实施方式，其中，所述GPx7基因是指内质网谷胱甘肽过氧化物酶7基因，其NCBI Gene ID为2882。
根据本公开的第六种优选的实施方式，其中，所述模板DNA可以包括左同源臂序列、敲入片段和右同源臂序列；优选地，所述左同源臂序列和所述右同源臂序列之间可以插入有CMV增强子、CMV启动子、GPx7基因的表达框和bGH poly A；优选地，所述左同源臂序列和所述右同源臂序列之间插入的序列如SEQ ID NO.117所示。
根据本公开的第六种优选的实施方式，其中，优选地，所述sgRNA如SEQ ID NO.114所示；相应地，所述左同源臂序列如SEQ ID NO.115所示；相应地，所述右同源臂序列如SEQ ID NO.116所示。
根据本公开的第六种优选的实施方式，其中，可选地，所述载体的骨架序列如SEQ ID NO.120所示。所述载体的骨架序列是指未插入模板DNA的载体的序列。
根据本公开的第六种优选的实施方式，其中，可选地，扩增并定向诱导分化的条件包括：将所述进行了基因定向敲入的单克隆细胞株在低粘附培养板中继续培养18-100小时后形成拟胚体；然后将所述拟胚体接种于基质胶上继续培养10-20天后用流式细胞术分选其中CD73、CD90和CD105均为阳性的细胞群体。
以下通过实施例进一步详细说明本发明：
实施例1-1，用于详细说明本公开的第一种优选实施方式。
1、sgRNA设计以及合成
(1)通过在线sgRNA设计工具(http://crispr.mit.edu/)在HBB基因一号外显子上设计靶向识别的HBB sgRNA。
HBB sgRNA:5’-cuugccccacagggcaguaaguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu-3’(SEQ ID NO.1)。
在HBB sgRNA中1-20位的序列为识别基序，其余的序列为tracrRNA。
(2)HBB sgRNA由Integrated DNA Technologies US合成并在HBB sgRNA序列的5’端和3’端末尾分别添加O-Me、phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)从基因组扩增识别靶序列片段(2400bp)，引物由生工生物工程(上海)股份有限公司合成。
HBB-FW:5’-tagatgtccccagttaacctcctat-3’(SEQ ID NO.2)，
HBB-REV:5’-ttattaggcagaatccagatgctca-3’(SEQ ID NO.3)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2μL配置成20μL反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为人诱导多能干细胞1016细胞株，根据AAV组织亲和性对照表，优选血清型为AAV-DJ的包装系统。
(2)AAV-DJ的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。
左同源臂序列为SEQ ID NO.4，其中第495-497位碱基为经过突变的PAM位点。
右同源臂序列为SEQ ID NO.5。
插入片段为一个完整的Cassette包括CMV增加子、CMV启动子、EGFP和bGH poly A，共约1452bp，如SEQ ID NO.6所示，其中第1-507位为启动子，第508-1227位为EGFP，第1228-1452位为bGH polyA。使用NheI和BsmI限制性内切酶(购于NEB(北京)有限公司)将Cassette插入到pAAV载体上，载体序列为SEQ ID NO.7。为了避免SpCas9/sgRNA RNP复合物对重组位点的二次切割从而影响基因编辑的效率，通过PrimerStar高保真DNA聚合酶(购于宝生物工程(大连)有限公司，货号：R044A)利用PCR刻环的方法在PAM位点引入了点突变(CGG突变为CTG)，从而避免了了SpCas9/sgRNA RNP复合物对识别位点的切割。点突变引物由生工生物工程(上海)股份有限公司合成。
HBB-PAM-mut-FW:5’-cctgaggagaagtctgcagttactgccctgtgggg-3’(SEQ ID NO.8)。
HBB-PAM-mut-REV:5’-ccccacagggcagtaactgcagacttctcctcagg-3’(SEQ ID NO.9)。
4、AAV病毒包装及纯化
(1)在病毒包装前一天将HEK293T细胞按照每皿5×10 6的数量种植到直径10cm的含有10mL完全培养基(DMEM+10％FBS+1％P/S双抗)(DMEM培养基，购于ThermoFisher Scientific,Inc.，货号：C11995500BT；FBS，购于ThermoFisher Scientific,Inc.，货号：sv30087.02；P/S双抗，购于ThermoFisher Scientific,Inc.，货号：SV30010)的CORNING培养皿中，共种植30皿。在37度、5％CO 2的细胞培养箱中培养24小时。
(2)病毒包装当天检查HEK293T细胞汇合度是否达到80％。每皿按照以下步骤单独配置转染体系：将10μg的pAAV-Cassette，10μg的pHelper，10μg的pAAV-DJ-RC混合后使用无血清的DMEM(购于购于ThermoFisher Scientific,Inc.，货号：11320082)调整体积到910μL后经漩涡振荡器混匀，加入90μL的PEI(购于Polysciences Asia Pacific,Inc.货号：23966-2)后再次使用漩涡振荡器混匀，静置15分钟。将CORNING培养皿中的完全培养基替换成9mL无血清培养基，再将之前经过静置的DNA-PEI复合物均匀滴加到培养皿中，轻柔晃匀后置于37度、5％CO 2的细胞培养箱中培养6小时。转染6h后将CORNING培养皿中的无血清培养基替换成完全培养基，于37度、5％CO 2的细胞培养箱中继续培养。
(3)在转染60小时之后，分别收获上清和细胞。
将收集得到的上清经4000rpm 4度离心10分钟后弃去杂质。将去除杂质的上清加入Amicon Ultra-15超离柱中(购于默克化工技术(上海)有限公司，货号：UFC 905096)，经过若干次4000rpm、4度离心30分钟将体积浓缩至10到15mL。将用细胞刮刀刮下的HEK293T细胞用适量培养基吹匀并转移至50mL离心管中，经1500rpm、4度离心10min后弃上清，所有沉淀总共加3mL细胞裂解缓冲液(150mM NaCl,20mM tris pH8.0)使其重悬。将重悬细胞在-80℃酒精浴和37℃水浴中反复冻融三次。将浓缩的上清和冻融的细胞悬液混匀，添加1M MgCl2至终浓度为1mM。添加Benzonase(购于默克化工技术(上海)有限公司，货号：70746-1kU)至终浓度为25U/mL，混匀后37℃反应40min。取出50mL离心管，4℃，4000rpm离心20min，取上清。
(4)采用碘克沙醇密度梯度离心法纯化病毒(购于Sigma-Aldrich，货号：D1556-250mL)。配置碘克沙醇梯度17％：5mL 10×PBS,0.05mL 1M MgCl 2,0.125mL 1M KCl,10mL 5M NaCl,12.5mL Optiprep，加水补足到50mL。25％：5mL 10×PBS,0.05mL 1M MgCl2,0.125mL 1M KCl,20mL Optiprep,0.1mL 0.5％phenol red，加水补足到50mL。40％：5mL 10×PBS,0.05mL 1M MgCl 2,0.125mL 1M KCl,33.3mL Optiprep，加水补足到50mL。60％：0.05mL 1M MgCl 2,0.125mL 1M KCl,50mL Optiprep,0.025mL 0.5％phenol red。向超速离心管中由下至上依次缓慢加入3.5mL 60％、3.5mL40％、4mL 25％、4mL 17％的碘克沙醇。将浓缩的上清和细胞裂解液缓慢加在离心管最上层，用细胞裂解缓冲液补满离心管。使用贝克曼L-80XP落地超速离心机、70Ti定角转子，加速6，减速9，60000rpm 4度离心2小时。用平头注射器吸取40％浓度层碘克沙醇，转移至Amicon Ultra-15超离柱中，加入10mL PBS 4000rpm 4℃离心20分钟，重复3次。将病毒离心浓缩至1mL。
(5)利用qPCR对AAV-DJ进行滴度检测。根据EGFP序列设计引物，使qPCR产物的长度约200bp。qPCR引物由生工生物工程(上海)股份有限公司合成。
AAVGFPF:5’-tcagcttcaggcaccaccac-3’(SEQ ID NO.10)。
AAVGFPR:5’-tgaacttgtggccgtttacgtcg-3’(SEQ ID NO.11)。
制备7个AAV-DJ重组质粒标准品，以1：10的比例进行梯度稀释。从10ng/μL以下的浓度开始稀释，分别为10ng/μL、1ng/μL、0.1ng/μL、0.01ng/μL、0.001ng/μL、0.0001ng/μL和0.00001ng/μL。
根据以下公式计算稀释的DNA拷贝数：
Figure PCTCN2018119852-appb-000005
使用DNaseI对病毒样本进行预处理(购于宝生物工程(大连)有限公司，货号：2270A)。使用2×SYBR PCR mix(购于东洋纺(上海)生物科技有限公司，货号：QPS-201)配置qPCR反应体系。使用Roche
Figure PCTCN2018119852-appb-000006
480II实时荧光定量PCR系统进行定量PCR。根据Ct值，绘制标准曲线，并计算AAV-DJ的滴度。
5、RNP复合物组装及待编辑细胞的预处理
(1)将SpCas9(终浓度1μmol/μL)和HBB sgRNA(终浓度1μmol/μL)按照1:3的摩尔比进行混合在室温孵育10min，从而形成SpCas9/sgRNA RNP复合物。
(2)观察1016细胞株(购自美国哈佛大学干细胞库)汇合率达到80％后移除mTeSR培养基(购于Stem Cell Technologies,Inc.，货号：85850)，用PBS漂洗一次。加入Accutase消化酶(购于ThermoFisher Scientific,Inc.，货号：A1110501)使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去Accutase消化酶。即刻加入新鲜的mTeSR培养基，用1mL枪扇形吹打培养皿底，使皿/瓶底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，制成干细胞悬液。使用血球计数板对细胞密度进行计数。使用Opti-MEM(14.5mM的ATP、23.6mM的氯化镁)(购于ThermoFisher Scientific,Inc.，货号：11058021)调整细胞密度到5×10 7/mL。
(3)将10μL经过室温孵育的SpCas9/sgRNA SNP复合物(以sgRNA的量计，所述RNP复合物的含量为7.5μmol)和10μL经过计数并使用Opti-MEM重悬的1016细胞悬液(其中细胞数量为5×10 5个)相混合，将20μL混合液转移到16孔电转板条中。使用Lonza 4D核转染系统选择CB150模式(电场强度为150V/cm，单次脉冲时间为10ms，相邻两次脉冲之间的时间间隔为20s，总脉冲次数为5次)进行电转。电转完成后立刻将细胞转移到经过Geltrex包被(购于ThermoFisher Scientific,Inc.，货号：A1413202)并且添加了500μL mTeSR和10μM Y-27632(购于Stem Cell Technologies,Inc.，货号：72304)的24孔板中于37度、5％CO 2的细胞培养箱中继续培养。
(4)电转完第5到20分钟内开始按照1×10 5的MOI值轻柔地滴加AAV-DJ，并于电转完20分钟内滴加完毕。
(5)电转完24小时后将旧培养基移除，更换为新的mTeSR培养基(含有10μM的Y-27632)
6、单克隆细胞株的培养：
(1)电转完第48小时使用PBS漂洗一次培养皿。加入Accutase消化酶使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去Accutase消化酶。即刻加入新鲜的mTeSR培养基，用1mL枪扇形吹打培养皿/瓶底，使皿底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，保证细胞间没有粘连，制成干细胞悬液。使用血球计数板对细胞密度进行计数。按每个10cm培养皿15000个细胞的比例把细胞种植到经过Geltrex包被并且添加了10mL mTeSR和10μM Y-27632的10cm CORNING培养皿中于37度、5％CO 2的细胞培养箱中继续培养。
(2)每24小时进行观察并移除旧的培养基，更换为新的mTeSR培养基(含有10μM的Y-27632)。72小时后可以在镜下观察到克隆的形成。
(3)电转后12到14天在十倍的物镜下可以看到克隆的大小已经相当于一个硬币。不能让克隆继续变大或者相交。在经过Geltrex包被的96孔板上的每个孔中加入120μL mTeSR(含有10μM Y-27632)，标记板O。在超净台中通过显微镜进行观察，将P200移液器调节至45μL使用带有滤芯的枪头刮碎克隆，用移液器收集细胞并转移到96孔板的小孔中。
(4)克隆挑取之后每24小时后将旧的培养基移除，更换为新的mTeSR培养基(含有10μM的Y-27632)四天后细胞汇合率达到80％。
(5)在两块经过Geltrex包被的96孔板上的每个孔中加入133μL mTeSR(含有10μM Y-27632)，分别标记为板A、板B。将板O的每孔使用150μL的PBS漂洗。每孔中加入35μL的Accutase消化酶，消化5到10分钟后每孔中添加165μL的mTeSR(含有10μM Y-27632)。从孔中分别移取66μL的细胞悬液转移到板A和板B的对应小孔中。板A用于基因组提取，板B备用。在板O的每个孔中直接添加165μL的mFreSR(购于Stem Cell Technologies,Inc.)后至于深低温冰箱存储。
7、基因编辑效率检测(等位基因插入检测)
(1)因为在HBB基因座上插入的Cassette可以表达绿色荧光蛋白，所以使用流式细胞仪检测 并统计能发出绿色荧光的细胞数量以及细胞总量。将能发出绿色荧光的细胞数量除以细胞总量可以的到总的基因编辑效率。
(2)在同源臂外设计引物(非整合位点PCR产物约2400bp左右，整合位点PCR产物3900bp)。引物由生工生物工程(上海)股份有限公司合成。
HBB-FW：5’-tagatgtccccagttaacctcctat-3’(SEQ ID NO.12)，
HBB-REV：5’-ttattaggcagaatccagatgctca-3’(SEQ ID NO.13)。
(3)选取20个克隆以及未编辑1016细胞株作为对照，将A板或B板96孔板中的克隆所获得的基因组进行PCR扩增。
(4)将PCR产物进行电泳并分析。只有2400bp左右条带的为非编辑细胞，只有3900bp条带的为双等位基因编辑细胞，既有2400bp左右条带也有3900bp条带的为单等位基因编辑细胞。分别标注并统计这三类编辑类型的比例。
对比例1-1
按照实施例1-1的方法进行，不同的是电转完第35min开始按照1×10 5的MOI轻柔地滴加AAV-DJ，并于电转完50分钟内滴加完毕。
测试实施例1-1
1、荧光检测等位基因插入效率检测
检测样品：经过电转和AAV感染后第四天经过Accutase消化酶后加入新鲜的mTeSR培养基使其重悬制成的干细胞悬液。
检测方法：因为在目标基因座上插入的Cassette可以表达绿色荧光蛋白，使用流式细胞仪检测并统计能发出绿色荧光的细胞数量以及细胞总量。将能发出绿色荧光的细胞数量除以细胞总量可以得到总的编辑效率。
实验结果：实施例1-1编辑效率为81.2％，对比例1-1编辑效率为10.6％。
2、PCR检测等位基因插入效率
检测样品：96孔板培养的单克隆细胞株(A板和B板)
检测方法：在同源臂外设计引物(非整合位点PCR产物约2400bp左右，整合位点PCR产物4600bp)。引物由生工生物工程(上海)股份有限公司合成。
实施例1-1、对比例1-1使用HBB-FW：5’-tagatgtccccagttaacctcctat-3’(SEQ ID NO.12)
HBB-REV：5’-ttaggcagaatccagatgctca-3’(SEQ ID NO.13)。
将96孔板中的克隆所获得的基因组进行PCR扩增。将PCR产物进行电泳并分析。只有2400bp左右条带的为非编辑细胞，只有4600bp的为双等位基因编辑细胞，既有2400bp左右条带也有4600bp的为单等位基因编辑细胞。
实验结果：实施例1-1编辑效率：等位基因双编辑64.2％、等位基因单编辑16.4％，对比例1-1编辑效率：等位基因双编辑0.3％、等位基因单编辑0.7％；可见电转与病毒转染的时间间隔对基因敲入的效率影响较大，在优选在电转结束后5-20分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行8-24小时的转染的情况下，基因敲入的效率最高。
实施例2-1，用于详细说明本公开的第二种优选实施方式。
构建在间充质干细胞Klotho基因中插入EGFP报告基因的细胞模型。采用Klotho基因原位基因启动子，在终止密码子前插入T2A-EGFP。
1、sgRNA设计以及合成
(1)通过在线sgRNA设计工具(http://crispr.mit.edu/)针对人源Klotho基因的TAG终止子上游100bp以内的序列设计靶向识别的sgRNA。
优选结果为：5’-ucacacccgaaagucuuuacguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaac uugaaaaaguggcaccgagucggugcuuuuuuu-3’(SEQ ID NO.14)。
其中1-20位的序列为识别基序，其余的序列为tracrRNA。
(2)该sgRNA由Integrated DNA Technologies公司合成并在该sgRNA序列的5’端和3’端末尾分别添加O-Me、phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)从基因组扩增识别靶序列片段(2786bp)，引物由生工生物工程(上海)股份有限公司合成。
Klotho-FW:5’-cataaagatttaaccttgctg-3’(SEQ ID NO.41)，
Klotho-REV:5’-gtatgtcattaaccagatacat-3’(SEQ ID NO.42)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2μL配置成20μL反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为人骨髓间充质干细胞，根据AAV组织亲和性对照表，优选血清型为AAV-9的包装系统。
(2)AAV-9的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。
左同源臂序列为SEQ ID NO.15，其中，第412-414位碱基为经过突变的PAM位点。
右同源臂序列为SEQ ID NO.16。
插入片段为T2A和EGFP，共777bp，序列如SEQ ID NO.43所示。使用无缝克隆(NEBuilder高保真DNA组装试剂盒，购于NEB(北京)有限公司，货号：E2621S)的方法将Cassette插入到pAAV载体上，载体序列为SEQ ID NO.44。为了避免SpCas9/sgRNA RNP复合物对重组位点的二次切割从而影响基因编辑的效率，通过PrimerStar高保真DNA聚合酶(购于宝生物工程(大连)有限公司，货号：R044A)利用PCR刻环的方法在PAM位点引入了点突变(TGG突变为TCG)，从而避免了SpCas9/sgRNA RNP复合物对识别位点的切割。点突变引物由生工生物工程(上海)股份有限公司合成。
Klotho-PAM-mut-FW:5’-acccgaaagtctttactcgctttcatagcttttct-3’(SEQ ID NO.45)。
Klotho-PAM-mut-REV:5’-agaaaagctatgaaagcgagtaaagactttcgggt(SEQ ID NO.46)。
4、AAV病毒包装及纯化
(1)在病毒包装前一天将HEK293T细胞按照每皿5×10 6的数量种植到直径10cm的含有10mL完全培养基(DMEM(购于ThermoFisher Scientific,Inc.，货号：C11995500BT)+10％FBS(购于ThermoFisher Scientific,Inc.，货号：SV30087.02)+1％PS双抗购于ThermoFisher Scientific,Inc.，货号：SV30010)的CORNING培养皿中，共种植30皿。在37度、5％CO 2的细胞培养箱中培养24小时。
(2)病毒包装当天检查HEK293T细胞汇合度是否达到80％。每皿按照以下步骤单独配置转染体系：将10μg的pAAV-Cassette，10μg的pHelper，10μg的pAAV9-RC混合后使用无血清的DMEM调整体积到910μL后经漩涡振荡器混匀，加入90μL的PEI(购于Polysciences Asia Pacific,Inc.货号：23966-2)后再次使用漩涡振荡器混匀，静置15分钟。将CORNING培养皿中的完全培养基替换成9mL无血清培养基，再将之前经过静置的DNA-PEI复合物均匀滴加到培养皿中，轻柔晃匀后置于37 度、5％CO 2的细胞培养箱中培养6小时。转染6h后将CORNING培养皿中的无血清培养基替换成完全培养基，于37度、5％CO 2的细胞培养箱中继续培养。
(3)在转染60小时之后，分别收获上清和细胞。
将收集得到的上清经4000rpm 4度离心10分钟后弃去杂质。将去除杂质的上清加入Amicon Ultra-15超离柱中(购于默克化工技术(上海)有限公司，货号：UFC 905096)，经过若干次4000rpm、4度离心30分钟将体积浓缩至10到15mL。将用细胞刮刀刮下的HEK293T细胞用适量培养基吹匀并转移至50mL离心管中，经1500rpm、4度离心10min后弃上清，所有沉淀总共加3mL细胞裂解缓冲液(150mM NaCl,20mM tris pH8.0)使其重悬。将重悬细胞在-80℃酒精浴和37℃水浴中反复冻融三次。将浓缩的上清和冻融的细胞悬液混匀，添加1M MgCl 2至终浓度为1mM。添加Benzonase(购于默克化工技术(上海)有限公司，货号：70746-1kU)至终浓度为25U/mL，混匀后37℃反应40min。取出50mL离心管，4℃，4000rpm离心20min，取上清。
(4)采用如上相同的碘克沙醇密度梯度离心法纯化病毒。
(5)利用qPCR对AAV9进行滴度检测。根据EGFP序列设计引物，使qPCR产物的长度约200bp。qPCR引物由生工生物工程(上海)股份有限公司合成。
AAVGFPF:5’-tcagcttcaggcaccaccac-3’(SEQ ID NO.47)。
AAVGFPR:5’-tgaacttgtggccgtttacgtcg-3’(SEQ ID NO.48)。
5、RNP复合物组装及待编辑细胞的预处理
(1)将SpCas9(终浓度300ug/ml)和Klotho sgRNA(终浓度175ug/ml)按照1:3的摩尔比进行混合在室温孵育10min，从而形成SpCas9/sgRNA RNP复合物。
(2)骨髓间充质干细胞汇合率达到80％后，制成干细胞悬液，调整细胞密度到5×10 7/mL。
(3)将10μL经过室温孵育的SpCas9/sgRNA SNP复合物(以sgRNA的量计，所述RNP复合物的含量为7.5μmol)和10μL经过计数并使用Opti-MEM重悬的骨髓间充质干细胞悬液(其中细胞数量为5×10 5个)相混合，将20μL混合液转移到16孔电转板条中。使用Lonza 4D核转染系统选择CB150模式(电场强度为150V/cm，单次脉冲时间为10ms，相邻两次脉冲之间的时间间隔为20s，总脉冲次数为5次)进行电转。电转完成后立刻将细胞转移到含DMEM/F12完全培养基的24孔板中于37度、5％CO 2的细胞培养箱中继续培养。
(4)电转完第5到20分钟内开始按照1×10 5的MOI值轻柔得滴加AAV9，并于电转完20分钟内滴加完毕。
(5)电转完24小时后将旧培养基移除，更换为DMEM/F12完全培养基。
6、单克隆细胞株的培养：
(1)电转完第48小时使用PBS漂洗一次培养皿。加入胰酶使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去胰酶。即刻加入新鲜的DMEM/F12完全培养基，用1mL枪扇形吹打培养皿/瓶底，使皿底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，保证细胞间没有粘连，制成干细胞悬液。使用血球计数板对细胞密度进行计数。按每个10cm培养皿15000个细胞的比例把细胞种植到含DMEM/F12完全培养基的10cm CORNING培养皿中于37度、5％CO 2的细胞培养箱中继续培养。
(2)每24小时进行观察并移除旧的培养基，更换为新的DMEM/F12完全培养基。72小时后可以在镜下观察到克隆的形成。
(3)电转后12到14天在十倍的物镜下可以看到克隆的大小已经相当于一个硬币。不能让克隆继续变大或者相交。在96孔板上的每个孔中加入120μL DMEM/F12完全培养基，标记板O。在超净台中通过显微镜进行观察，将P200移液器调节至45μL使用带有滤芯的枪头刮碎克隆，用移液器收集细胞并转移到96孔板的小孔中。
(4)克隆挑取之后每24小时后将旧的培养基移除，更换为新的DMEM/F12完全培养基，四天 后细胞汇合率达到80％。
(5)在两块96孔板上的每个孔中加入133μL DMEM/F12完全培养基，分别标记为板A、板B。将板O的每孔使用150μL的PBS漂洗。每孔中加入35μL的胰酶，消化5到10分钟后每孔中添加165μL的DMEM/F12完全培养基。从孔中分别移取66μL的细胞悬液转移到板A和板B的对应小孔中。板A用于基因组提取，板B备用。在板O的每个孔中直接添加165μL的细胞冻存液后至于深低温冰箱存储。
实施例2-2，用于详细说明本公开的第二种优选实施方式。
构建在骨髓间充质干细胞周期调控基因p16基因中插入EGFP报告基因的细胞模型。采用p16基因原位基因启动子，在终止密码子前插入2A-EGFP。
通过在线sgRNA设计工具(http://crispr.mit.edu/)在p16基因第2个外显子靠近终止密码子(TGA)上游100bp以内的序列设计靶向识别的sgRNA。
p16sgRNA：5’-gggccgucugcccguggaccguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu-3’。(SEQ ID NO.17)。
其中1-20位的序列为识别基序，其余的序列为tracrRNA。
该sgRNA委托Integrated DNA Technologies公司合成，并对临近该sgRNA的5’和3’末端分别进行O-methyl及-phosphorothioate修饰。
2、体外法检测sgRNA的活性
(1)从基因组扩增识别靶序列片段(1084bp)，引物由生工生物工程(上海)股份有限公司合成。
P16-FW:5’-tgaagttcaacattcccagaagc-3’，(SEQ ID NO.49)，
P16-REV:5’-agggtcagcgaagtcttggt-3’，(SEQ ID NO.50)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2ul配置成20ul反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为脂肪间充质(ADSC)干细胞株，根据AAV组织亲和性对照表，优选血清型为AAV9的包装系统。
(2)AAV9的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。左同源臂序列为SEQ ID NO.18，其中第489-494位碱基为经过突变的位于PAM位点上游的第2,3个密码子，因为PAM位点无法通过同义突变而失效，右同源臂序列为SEQ ID NO.19。插入片段为T2A和EGFP，共777bp，序列如SEQ ID NO.43所示。使用无缝克隆(NEBuilder高保真DNA组装试剂盒，购于NEB(北京)有限公司，货号：E2621S)的方法将Cassette插入到pAAV载体上，载体序列为SEQ ID NO.44。为了避免SpCas9/sgRNA RNP复合物对重组位点的二次切割从而影响基因编辑的效率，通过PrimerStar高保真DNA聚合酶(购于宝生物工程(大连)有限公司，货号：R044A)利用PCR刻环的方法在位于PAM位点‘上游的第2、3个密码子引入点突变(CGT变为CGC，GGA变为GGC)，从而避免了SpCas9/sgRNA RNP复合物对识别位点的切割。点突变引物由生工生物工程(上海)股份有限公司合成。
P16-PAM-mut-FW:5’-cgcgatgcctggggccgtctgcccgcggccctggc-3’(SEQ ID NO.51)。
P16-PAM-mut-REV:5’-gccagggccgcgggcagacggccccaggcatcgcg-3’(SEQ ID NO.52)。
4、AAV病毒包装及纯化
同实施例2-1
5、RNP复合物组装及待编辑细胞的预处理
同实施例2-1
6、单克隆细胞株的培养
同实施例2-1
对比例2-1
按照实施例2-1的方法进行，不同的是电转完第35min开始按照1×10 5的MOI轻柔得滴加AAV9，并于电转完50分钟内滴加完毕。
测试实施例2-1
1、荧光检测等位基因插入效率检测
检测样品：实施例2-1、2-2以及对比例2-1经过电转和AAV感染后第四天经过胰酶消化后加入新鲜的DMEM/F12完全培养基使其重悬制成的干细胞悬液。
检测方法：因为在目标基因座上插入的T2A-EGFP序列表达绿色荧光蛋白，使用流式细胞仪检测并统计能发出绿色荧光的细胞数量以及细胞总量。将能发出绿色荧光的细胞数量除以细胞总量可以的到总的编辑效率。
实验结果：实施例2-1编辑效率为73％，实施例2-2编辑效率为76％，对比例2-1编辑效率为8％。
2、PCR检测等位基因插入效率
检测样品：96孔板培养的单克隆细胞株(A板和B板)
检测方法：在同源臂外设计引物。引物由生工生物工程(上海)股份有限公司合成。
实施例2-1、对比例2-1使用Klotho-FW、Klotho-REV。实施例2-2使用p16-FW、p16-REV。
将96孔板中的克隆所获得的基因组进行PCR扩增。将PCR产物进行电泳并分析。实施例2-1、对比例2-1只有2786bp左右条带的为非编辑细胞，只有3563bp的为双等位基因编辑细胞，既有2786bp左右条带也有3563bp的为单等位基因编辑细胞。实施例2-2中只有1413bp左右条带的为非编辑细胞，只有2190bp的为双等位基因编辑细胞，既有1413bp左右条带也有2190bp的为单等位基因编辑细胞。
实验结果：实施例2-1编辑效率：等位基因双编辑62％、等位基因单编辑77％，对比例2-1编辑效率：等位基因双编辑1％、等位基因单编辑7％；实施例2-2编辑效率：等位基因双编辑58％、等位基因单编辑76％；可见电转与病毒转染的时间间隔对基因敲入的效率影响较大，在优选在电转结束后5-20分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行8-24小时的转染的情况下，基因敲入的效率最高。
3、所构建细胞模型在体外压力下连续传代所致的细胞衰老检测
将实施例2-1得到的进行了EGFP敲入的骨髓间充质干细胞的骨髓间充质干细胞分别铺种于6孔板，贴壁后，分别加入终浓度为100μM的DNA损伤诱导剂4-硝基喹啉-N-氧化物(购于Sigma-Aldrich，货号：N8141)以及终浓度为6μg/mL的内质网应激诱导剂——衣霉素(购于Sigma-Aldrich，货号：654380-10MG)，总体积为2mL，处理两组。依次收集处理0h、12h、24h、48h、72h的细胞后，一组用流式细胞仪检测样本EGFP的发光情况，统计发光细胞所占百分比；另一组用β-半乳糖苷酶染色试剂盒(购于北京索莱宝科技有限公司，货号：G1580)进行染色，然后在光学显微镜下观察X-gal的显色情况，统计显色细胞所占百分比。结果如表2-2(4-硝基喹啉-N-氧化 物)，表2-3(衣霉素)所示。
表2-2 DNA损伤压力下细胞衰老状态检测(实施例2-1)
  0h 12h 24h 48h 72h
EGFP(荧光表达细胞比率) 97％ 85％ 73％ 54％ 16％
X-gal(阳性细胞比率) 0％ 12％ 30％ 69％ 97％
表2-3内质网损伤压力下细胞衰老状态检测(实施例2-1)
  0h 12h 24h 48h 72h
EGFP(荧光表达细胞比率) 96％ 89％ 75％ 60％ 32％
X-gal(阳性细胞比率) 0％ 7％ 26％ 51％ 83％
上述表2-2和表2-3结果显示，实施例2-1中所建立的模型可在不致细胞死亡的情况下，实时检测不同压力下的细胞衰老，Klotho基因下调与细胞衰老相关，EGFP荧光表达细胞比率下降代表衰老细胞数量增多。与X-gal染色法相比，具有一致的细胞衰老趋势。
实施例2-2操作同实施例2-1，用4-硝基喹啉-N-氧化物或衣霉素处理后的细胞发光或显色百分比如表2-4、2-5所示。
表2-4 DNA损伤压力下细胞衰老状态检测(实施例2-2)
  0h 12h 24h 48h 72h
EGFP(荧光表达细胞比率) 0％ 9％ 26％ 67％ 94％
X-gal(阳性细胞比率) 0％ 14％ 31％ 71％ 97％
表2-5内质网损伤压力下细胞衰老状态检测(实施例2-2)
  0h 12h 24h 48h 72h
EGFP(荧光表达细胞比率) 0％ 13％ 23％ 68％ 89％
X-gal(阳性细胞比率) 0％ 11％ 28％ 70％ 90％
上述表2-4和表2-5结果显示，实施例2-2中所建立的模型可在不致细胞死亡的情况下，实时检测不同压力下的细胞衰老，p16基因上调与细胞衰老相关，EGFP荧光表达细胞比率上升代表衰老细胞数量增多。与X-gal染色法相比，具有一致的细胞衰老趋势。
4、所构建细胞模型在正常情况下连续传代所致的细胞衰老检测
将实施例1得到的进行了EGFP敲入的骨髓间充质干细胞的骨髓间充质干细胞铺种于10cm dish中正常传代，依次将0代，2代，5代，10代，15代细胞铺种于96孔中，利用CellTiter试剂盒(购于Promega，货号：G3582)检测，统计对应代数细胞的倍增活力以此表征细胞的衰老情况。同时取相应代数的细胞用流式细胞仪检测EGFP的发光情况，统计发光细胞所占的百分比。结果如表2-6所示。
表2-6连续传代下细胞衰老检测(实施例2-1)
  0代 2代 5代 10代 15代
CellTiter(细胞倍增速率) 100％ 96％ 60％ 25％ 11％
EGFP(荧光表达细胞比率) 99％ 98％ 87％ 75％ 32％
结果表明：在正常连续传代压力下，实施例2-1构建的细胞检测模型中EGFP荧光表达细胞比率的下降伴随着细胞活力下降，由此可以实时检测间充质干细胞衰老。
实施例2-2操作同实施例2-1，结果如表2-7。
表2-7连续传代下细胞衰老检测(实施例2-2)
  0代 2代 5代 10代 15代
CellTiter(细胞倍增速率) 100％ 95％ 73％ 21％ 9％
EGFP(荧光表达细胞比率) 1％ 15％ 23％ 47％ 89％
结果表明：在正常连续传代压力下，实施例2-2构建的细胞检测模型中EGFP荧光表达细胞比率的上升伴随着细胞活力下降，由此可以实时检测间充质干细胞衰老。
实施例3-1，用于详细说明本公开的第三种优选实施方式。
构建在脐带间充质干细胞程序性死亡受体1基因(PD-1)的Exon1中插入膜结合型TRAIL基因表达Cassette的细胞模型。
1、sgRNA设计以及合成
(1)通过在线sgRNA设计工具(http://crispr.mit.edu/)在PD-1基因1号外显子上设计靶向识别的PD-1sgRNA，优选结果如下：5’-
cgacuggccagggcgccuguguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu-3’(SEQ ID NO.53)。
其中1-20位的序列为识别基序，其余的序列为tracrRNA。
(2)该sgRNA由Integrated DNA Technologies,Inc.(IDT)合成并在该sgRNA序列的5’端和3’端末尾的三个碱基的二号位和三号位上别添加O-Me、phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)从基因组扩增识别靶序列片段(1456bp)，引物由生工生物工程(上海)股份有限公司合成。
PD-1-FW:5’-ggaaagaggccacagcagtg-3’(SEQ ID NO.57)，
PD-1-REV:5’-agtcgcctgccacagtgaag-3’(SEQ ID NO.58)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2μL配置成20μL反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为脐带间充质干细胞，根据AAV组织亲和性对照表，优选血清型为AAV-9的包装系统。
(2)AAV-9的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。
左同源臂序列为SEQ ID NO.54。
右同源臂序列为SEQ ID NO.55。
插入片段一个完整的Cassette包括CMV增加子、CMV启动子、膜结合型TRAIL CDS和SV40poly A，共1487bp，序列如SEQ ID NO.56所示。使用无缝克隆(NEBuilder高保真DNA组装试剂盒，购于NEB(北京)有限公司，货号：E2621S)的方法将Cassette插入到pAAV载体上，载体序列为SEQ ID NO.59。
4、AAV病毒包装及纯化
(1)在病毒包装前一天将HEK293T细胞按照每皿5×10 6的数量种植到直径10cm的含有10mL完全培养基：DMEM(购于ThermoFisher Scientific,Inc.，货号：C11995500BT)+10％FBS(购于ThermoFisher Scientific,Inc.，货号：sv30087.02)+1％P/S双抗(购于ThermoFisher Scientific,Inc.，货号：SV30010)的CORNING培养皿中，共种植30皿。在37度、5％CO 2的细胞培养箱中培养24小时。
(2)病毒包装当天检查HEK293T细胞汇合度是否达到80％。每皿按照以下步骤单独配置转染体系：将10μg的pAAV-Cassette，10μg的pHelper，10μg的pAAV9-RC混合后使用无血清的DMEM调整体积到910μL后经漩涡振荡器混匀，加入90μL的PEI(购于Polysciences Asia Pacific,Inc.货号：23966-2)后再次使用漩涡振荡器混匀，静置15分钟。将CORNING培养皿中的完全培养基替换成9mL无血清培养基，再将之前经过静置的DNA-PEI复合物均匀滴加到培养皿中，轻柔晃匀后置于37度、5％CO 2的细胞培养箱中培养6小时。转染6h后将CORNING培养皿中的无血清培养基替换成完全培养基，于37度、5％CO 2的细胞培养箱中继续培养。
(3)在转染60小时之后，分别收获上清和细胞。
将收集得到的上清经4000rpm 4度离心10分钟后弃去杂质。将去除杂质的上清加入Amicon Ultra-15超离柱中(购于默克化工技术(上海)有限公司，货号：UFC 905096)，经过若干次4000rpm、4度离心30分钟将体积浓缩至10到15mL。将用细胞刮刀刮下的HEK293T细胞用适量培养基吹匀并转移至50mL离心管中，经1500rpm、4度离心10min后弃上清，所有沉淀总共加3mL细胞裂解缓冲液(150mM NaCl,20mM tris pH8.0)使其重悬。将重悬细胞在-80℃酒精浴和37℃水浴中反复冻融三次。将浓缩的上清和冻融的细胞悬液混匀，添加1M MgCl 2至终浓度为1mM。添加Benzonase(购于默克化工技术(上海)有限公司，货号：70746-1kU)至终浓度为25U/mL，混匀后37℃反应40min。取出50mL离心管，4℃，4000rpm离心20min，取上清。
(4)采用碘克沙醇密度梯度离心法纯化病毒。
(5)利用qPCR对AAV9进行滴度检测。根据EGFP序列设计引物，使qPCR产物的长度约200bp。qPCR引物由生工生物工程(上海)股份有限公司合成。
AAV-TRAIL-F:5’-gatcttcacagtgctcctgc-3’(SEQ ID NO.60)。
AAV-TRAIL-R:5’-tgacggagttgccacttgac-3’(SEQ ID NO.61)。
5、RNP复合物组装及待编辑细胞的预处理
(1)将SpCas9(终浓度300ug/ml)和PD1sgRNA(终浓度175μg/ml)按照1:3的摩尔比进行混合在室温孵育10min，从而形成SpCas9/sgRNA RNP复合物。
(2)脐带间充质干细胞汇合率达到80％后，制成干细胞悬液。使用血球计数板对细胞密度进行计数。使用Opti-MEM(14.5mM的ATP、23.6mM的氯化镁,购于ThermoFisher Scientific,Inc.，货号：11058021)调整细胞密度到5×10 7/mL。
(3)将10μL经过室温孵育的SpCas9/sgRNA SNP复合物(以sgRNA的量计，所述RNP复合物的含量为7.5μmol)和10μL经过计数并使用Opti-MEM重悬的骨髓间充质干细胞悬液(其中细胞数量为5×10 5个)相混合，将20μL混合液转移到16孔电转板条中。使用Lonza 4D核转染系统选择CB150模式(电场强度为150V/cm，单次脉冲时间为10ms，相邻两次脉冲之间的时间间隔为20s，总脉冲次数为5次)进行电转。电转完成后立刻将细胞转移到含添加了10％胎牛血清的DMEM/F12完全培养基的24孔板中于37度、5％CO 2的细胞培养箱中继续培养。
(4)电转完第5到20分钟内开始按照1×10 5的MOI值轻柔地滴加AAV9，并于电转完20分钟内滴加完毕。
(5)电转完24小时后将旧培养基移除，更换为DMEM/F12完全培养基。
6、单克隆细胞株的培养：
(1)电转完第48小时使用PBS漂洗一次培养皿。加入胰酶使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去胰酶。即刻加入新鲜的DMEM/F12完全培养基，用1mL枪扇形吹打培养皿/瓶底，使皿底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，保证细胞间没有粘连，制成干细胞悬液。使用血球计数板对细胞密度进行计数。按每个10cm培养皿15000个细胞的比例把细胞种植到含DMEM/F12完全培养基的10cm CORNING培养皿中于37度、5％CO 2的细胞培养箱中继续培养。
(2)每24小时进行观察并移除旧的培养基，更换为新的DMEM/F12完全培养基。72小时后可以在镜下观察到克隆的形成。
(3)电转后12到14天在十倍的物镜下可以看到克隆的大小已经相当于一个硬币。不能让克隆继续变大或者相交。在96孔板上的每个孔中加入120μL DMEM/F12完全培养基，标记板O。在超净台中通过显微镜进行观察，将P200移液器调节至45μL使用带有滤芯的枪头刮碎克隆，用移液器收集细胞并转移到96孔板的小孔中。
(4)克隆挑取之后每24小时后将旧的培养基移除，更换为新的DMEM/F12完全培养基，四天后细胞汇合率达到80％。
(5)在两块96孔板上的每个孔中加入133μL含10％胎牛血清的DMEM/F12完全培养基，分别标记为板A、板B。将板O的每孔使用150μL的PBS漂洗。每孔中加入35μL的胰酶，消化5到10分钟后每孔中添加165μL的DMEM/F12完全培养基。从孔中分别移取66μL的细胞悬液转移到板A和板B的对应小孔中。板A用于基因组提取，板B备用。在板O的每个孔中直接添加165μL的细胞冻存液后至于深低温冰箱存储。
实施例3-2，用于说明本公开的第三种优选实施方式。
构建在脐带间充质干细胞程序性死亡受体1基因(PD-1)的Exon1中插入分泌型TRAIL基因表达Cassette的细胞模型。
采用实施例3-1的方法进行，不同在于：插入片段为一个完整的Cassette包括CMV增加子、CMV启动子、重组分泌型TRAIL基因CDS和SV40poly A，共1349bp，序列如SEQ ID NO.62所示。
对比例3-1
按照实施例3-1的方法进行，不同的是电转完第35min开始按照1×10 5的MOI轻柔得滴加AAV9，并于电转完50分钟内滴加完毕。
对比例3-2
按照实施例3-2的方法进行，不同的是电转完第35min开始按照1×10 5的MOI轻柔得滴加AAV9，并于电转完50分钟内滴加完毕。
测试实施例3-1
1、基因编辑效率检测(等位基因插入检测)
(1)在同源臂外设计引物(非整合位点PCR产物约1341bp左右，针对实施例3-1和对比例3-1膜结合型TRAIL整合位点PCR产物2828bp，针对实施例3-2和对比例3-2分泌型TRAIL整合位点PCR产物2690bp)。引物由生工生物工程(上海)股份有限公司合成。
PD1-HR-FW:5’-ggagccgattagccatggac-3’(SEQ ID NO.63)，
PD1-HR-REV:5’-agaagaactgtcctcactcg-3’(SEQ ID NO.64)。
(2)选取96个克隆以及未编辑脐带间充质干细胞细胞株作为对照，将96孔板中的克隆所获得 的基因组进行PCR扩增。
(3)将PCR产物进行电泳并分析。只有1341bp左右条带的为非编辑细胞，只有2828bp的或2690bp的为双等位基因编辑细胞，既有1341bp左右条带也有2828bp的或2690bp的为单等位基因编辑细胞。分别标注并统计这三类编辑类型的比例。结果如表3-1所示。
表3-1
组别 非编辑细胞比例 双等位基因编辑细胞 单等位基因编辑细胞
实施例3-1 31.2％ 23.6％ 45.2％
对比例3-1 95.9％ 1.0％ 3.1％
实施例3-2 40.3％ 24.6％ 35.1％
对比例3-2 93.7％ 2.1％ 4.2％
2、实施例3-1和3-2得到脐带间充质干细胞对免疫抑制的解除作用(相对于未进行敲入的脐带间充质干细胞)：
细胞周期检查点(checkpoint)是细胞周期(cell cycle)中的一套保证DNA复制和染色体(chromosome)分配质量的检查机制，是一类负反馈调节机制。当细胞周期进程中出现异常事件，如DNA损伤或DNA复制受阻时，这类调节机制就被激活，及时地中断细胞周期的运行。待细胞修复或排除故障后，细胞周期才能恢复运转。将实施例3-1和3-2得到脐带间充质干细胞以及未进行敲入的脐带间充质干细胞分别移植入BALB/c小鼠，观测到实施例3-1和3-2得到脐带间充质干细胞对CD8 +T细胞抑制的解除(G0/G1期细胞数量减少，S期细胞数量增加)，结果如表3-2所示。
表3-2
组别 G0/G1期细胞比例 S期细胞比例
实施例3-1 21.6％ 67.8％
实施例3-2 23.1％ 65.9％
未敲入 68.4％ 21.2％
3、对荷瘤小鼠的肿瘤抑制实验：
参照文献《茶皂素对荷瘤小鼠肿瘤抑制作用研究》中的方法，制备人乳腺癌(EAC)荷瘤昆明小鼠模型(体重18～22g)，并用实施例3-1和3-2得到的脐带间充质干细胞和未进行敲入的脐带间充质干细胞对荷瘤小鼠分别进行尾静脉注射(注射剂量为1×10 6cell/0.1ml)，查看肿瘤抑制效果，结果如表3-3所示，其表明实施例3-1和3-2得到脐带间充质干细胞相对于未进行敲入的脐带间充质干细胞能够显著提高肿瘤抑制效率：(肿瘤对照组平均瘤重－实验组平均瘤重)/肿瘤对照组平均瘤重×100％。
表3-3
组别 平均瘤重(g) 肿瘤抑制效率
实施例3-1 0.94±0.13 58.4％
实施例3-2 1.01±0.21 55.3％
未敲入脐带间充质干细胞 1.92±0.46 15.0％
对照组 2.26±0.52 /
实施例4-1，用于详细说明本公开的第四种优选实施方式。
构建在脂肪间充质干细胞(ADSC细胞)骨桥蛋白基因(SPP1)中插入EGFP报告基因的细胞模型。采用SPP1基因原位基因启动子，在终止密码子前插入T2A-EGFP。
1、sgRNA设计以及合成
(1)通过在线sgRNA设计工具(http://crispr.mit.edu/)针对SPP1基因8号外显子靠近终止密码子(TAA)上游100bp以内的序列上设计靶向识别的sgRNA，优选结果如下：SPP1sgRNA:5’-gguuguagaccccaaaaguaguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu-3’(SEQ ID NO.65)。其中1-20位的序列为识别基序，其余的序列为tracrRNA。
(2)该sgRNA由Integrated Dna Technologies.USA公司合成并在该sgRNA序列的5’端和3’端末尾的三个碱基的二号位和三号位上分别添加O-Me、phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)基因组扩增识别靶序列片段(1144bp)，引物由生工生物工程(上海)股份有限公司合成。
SPP1-FW:5’-gtaacatgctagtattatttcagc-3’(SEQ ID NO.86)，
SPP1-REV:5’-aacaaaacatcacaccgtacc-3’(SEQ ID NO.87)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2μL配置成20μL反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为人脂肪间充质干细胞(ADSC细胞)，根据AAV组织亲和性对照表，优选血清型为AAV-9的包装系统。
(2)AAV-9的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。
左同源臂序列为SEQ ID NO.66，其中第433-435位碱基为经过突变的PAM位点。
右同源臂序列为SEQ ID NO.67。
插入片段为T2A和EGFP，共777bp，序列如SEQ ID NO.88所示。使用无缝克隆(
Figure PCTCN2018119852-appb-000007
高保真DNA组装试剂盒，购于NEB(北京)有限公司，货号：E2621S)的方法将Cassette插入到pAAV载体上，载体序列为SEQ ID NO.89。为了避免SpCas9/sgRNA RNP复合物对重组位点的二次切割从而影响基因编辑的效率，通过PrimerStar高保真DNA聚合酶(购于宝生物工程(大连)有限公司，货号：R044A)利用PCR刻环的方法在PAM位点引入了点突变(TGG突变为TCG)，从而避免了SpCas9/sgRNA RNP复合物对识别位点的切割。点突变引物由生工生物工程(上海)股份有限公司合成。
SPP1-PAM-mut-FW:5’-gtagaccccaaaagtaaagaagaagataaacacct-3’(SEQ ID NO.90)。
SPP1-PAM-mut-REV:5’-aggtgtttatcttctttacttttggggtctac-3’(SEQ ID NO.91)。
4、AAV病毒包装及纯化
(1)在病毒包装前一天将HEK293T细胞按照每皿5×10 6的数量种植到直径10cm的含有10mL完全培养基(DMEM+10％FBS+1％P/S双抗)(DMEM培养基，购于ThermoFisher Scientific,Inc.，货号：C11995500BT；FBS，购于ThermoFisher Scientific,Inc.，货号：sv30087.02；P/S双抗，购于ThermoFisher Scientific,Inc.，货号：SV30010)的CORNING培养皿中，共种植30皿。在37度、5％CO 2的细胞培养箱中培养24小时。
(2)病毒包装当天检查HEK293T细胞汇合度是否达到80％。每皿按照以下步骤单独配置转染体系：将10μg的pAAV-Cassette，10μg的pHelper，10μg的pAAV9混合后使用无血清的DMEM调整 体积到910μL后经漩涡振荡器混匀，加入90μL的PEI(购于Polysciences Asia Pacific,Inc.，货号：23966-2)后再次使用漩涡振荡器混匀，静置15分钟。将CORNING培养皿中的完全培养基替换成9mL无血清培养基，再将之前经过静置的DNA-PEI复合物均匀滴加到培养皿中，轻柔晃匀后置于37度、5％CO 2的细胞培养箱中培养6小时。转染6h后将CORNING培养皿中的无血清培养基替换成完全培养基，于37度、5％CO 2的细胞培养箱中继续培养。
(3)在转染60小时之后，分别收获上清和细胞。
将收集得到的上清经4000rpm 4度离心10分钟后弃去杂质。将去除杂质的上清加入Amicon Ultra-15超离柱中(购于默克化工技术(上海)有限公司，货号：UFC 905096)，经过若干次4000rpm、4度离心30分钟将体积浓缩至10到15mL。将用细胞刮刀刮下的HEK293T细胞用适量培养基吹匀并转移至50mL离心管中，经1500rpm、4度离心10min后弃上清，所有沉淀总共加3mL细胞裂解缓冲液(150mM NaCl,20mM tris pH8.0)使其重悬。将重悬细胞在-80℃酒精浴和37℃水浴中反复冻融三次。将浓缩的上清和冻融的细胞悬液混匀，添加1M MgCl 2至终浓度为1mM。添加Benzonase(购于默克化工技术(上海)有限公司，货号：70746-1kU)至终浓度为25U/mL，混匀后37℃反应40min。取出50mL离心管，4℃，4000rpm离心20min，取上清。
(4)采用碘克沙醇密度梯度离心法纯化病毒。
(5)利用qPCR对AAV9进行滴度检测。根据EGFP序列设计引物，使qPCR产物的长度约200bp。qPCR引物由生工生物工程(上海)股份有限公司合成。
AAVGFPF:5’-tcagcttcaggcaccaccac-3’(SEQ ID NO.92)。
AAVGFPR:5’-tgaacttgtggccgtttacgtcg-3’(SEQ ID NO.93)。
5、RNP复合物组装及待编辑细胞的预处理
(1)将SpCas9(终浓度300ug/ml)和SPP1sgRNA(终浓度175ug/ml)按照1:3的摩尔比进行混合在室温孵育10min，从而形成SpCas9/sgRNA RNP复合物。
(2)脂肪间充质干细胞汇合率达到80％后制成干细胞悬液，调整细胞密度到5×10 7/mL。
(3)将10μL经过室温孵育的SpCas9/sgRNA SNP复合物(以sgRNA的量计，所述RNP复合物的含量为7.5μmol)和10μL经过计数并使用Opti-MEM重悬的脂肪间充质干细胞悬液(其中细胞数量为5×10 5个)相混合，将20μL混合液转移到16孔电转板条中。使用Lonza 4D核转染系统选择CB150模式(电场强度为150V/cm，单次脉冲时间为10ms，相邻两次脉冲之间的时间间隔为20s，总脉冲次数为5次)进行电转。电转完成后立刻将细胞转移到含DMEM/F12完全培养基的24孔板中于37度、5％CO 2的细胞培养箱中继续培养。
(4)电转完第5到20分钟内开始按照1×10 5的MOI值轻柔地滴加AAV9，并于电转完20分钟内滴加完毕。
(5)电转完24小时后将旧培养基移除，更换为DMEM/F12完全培养基。
6、单克隆细胞株的培养：
(1)电转完第48小时使用PBS漂洗一次培养皿。加入胰酶使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去胰酶。即刻加入新鲜的DMEM/F12完全培养基，用1mL枪扇形吹打培养皿/瓶底，使皿底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，保证细胞间没有粘连，制成干细胞悬液。使用血球计数板对细胞密度进行计数。按每个10cm培养皿15000个细胞的比例把细胞种植到含DMEM/F12完全培养基的10cm CORNING培养皿中于37度、5％CO 2的细胞培养箱中继续培养。
(2)每24小时进行观察并移除旧的培养基，更换为新的DMEM/F12完全培养基。72小时后可以在镜下观察到克隆的形成。
(3)电转后12到14天在十倍的物镜下可以看到克隆的大小已经相当于一个硬币。不能让克隆继续变大或者相交。在96孔板上的每个孔中加入120μL DMEM/F12完全培养基，标记板O。在超净 台中通过显微镜进行观察，将P200移液器调节至45μL使用带有滤芯的枪头刮碎克隆，用移液器收集细胞并转移到96孔板的小孔中。
(4)克隆挑取之后每24小时后将旧的培养基移除，更换为新的DMEM/F12完全培养基，四天后细胞汇合率达到80％。
(5)在两块96孔板上的每个孔中加入133μL DMEM/F12完全培养基，分别标记为板A、板B。将板O的每孔使用150μL的PBS漂洗。每孔中加入35μL的胰酶，消化5到10分钟后每孔中添加165μL的DMEM/F12完全培养基。从孔中分别移取66μL的细胞悬液转移到板A和板B的对应小孔中。板A用于基因组提取，板B备用。在板O的每个孔中直接添加165μL的细胞冻存液后至于深低温冰箱存储。
实施例4-2，用于说明本公开的第四种优选实施方式。
构建在脂肪间充质干细胞(ADSC细胞)I型胶原蛋白基因(COL1A1)中插入EGFP报告基因的细胞模型。采用COL1A1基因原位基因启动子，在终止密码子前插入2A-EGFP。
通过在线sgRNA设计工具(http://crispr.mit.edu/)在COL1A1基因51号外显子靠近终止密码子(TAA)上游100bp以内的序列上设计靶向识别的sgRNA。
COL1A1 sgRNA：5’-uggggcaccaacguccaaggguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu-3’。(SEQ ID NO.68。
其中1-20位的序列为识别基序，其余的序列为tracrRNA。
该sgRNA由Integrated DNA Technologies.USA公司合成并在序列的5’端和3’端末尾的三个碱基的二号位和三号位上分别添加-2-O-Me、-3-phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)从基因组扩增识别靶序列片段(1084bp)，引物由生工生物工程(上海)股份有限公司合成。
COL1A1-FW:5’-ccctgcagttcgagtatgg-3’，(SEQ ID NO.94)，
COL1A1-REV:5’-gcagtctgagaaccccagg-3’，(SEQ ID NO.95)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2μl配置成20μl反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为脂肪间充质(ADSC)干细胞株，根据AAV组织亲和性对照表，优选血清型为AAV9的包装系统。
(2)AAV9的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。左同源臂序列为SEQ ID NO.69，其中第436-438位碱基为经过突变的PAM位点，右同源臂序列为SEQ ID NO.70。插入片段为T2A和EGFP，共777bp，序列如SEQ ID NO.88所示。使用无缝克隆(
Figure PCTCN2018119852-appb-000008
高保真DNA组装试剂盒，购于NEB(北京)有限公司,货号：E2621S)的方法将Cassette插入到pAAV载体上，载体序列为SEQ ID NO.89。为了避免SpCas9/sgRNA RNP复合物对重组位点的二次切割从而影响基因编辑的效率，通过PrimerStar高保真DNA聚合酶(购于宝生物工程(大连)有限公司，货号：R044A)利用PCR刻环的方法在PAM位点引入了点突变(TGG突变为TCG)，从而避免了SpCas9/sgRNA RNP复合物对识别位点的切割。点突变引物由生工生物工程(上海)股份有限公司合成。
COL1A1-PAM-mut-FW:5’-cccatcatcgatgtggcacccttggacgttggtgc-3’(SEQ ID NO.96)。
COL1A1-PAM-mut-REV:5’-gcaccaacgtccaagggtgccacatcgatgatggg-3’(SEQ ID NO.97)。
4、AAV病毒包装及纯化
同实施例4-1
5、RNP复合物组装及待编辑细胞的预处理
同实施例4-1
6、Lonza 4D系统电转及AAV感染
同实施例4-1
7、单克隆细胞株的培养
同实施例4-1
对比例4-1
按照实施例4-1的方法进行，不同的是电转完第35min开始按照1×10 5的MOI轻柔地滴加AAV9，并于电转完50分钟内滴加完毕。
测试实施例4-1
1、荧光检测等位基因插入效率检测
检测样品：实施例4-1、4-2以及对比例4-1经过电转和AAV感染后第四天经过胰酶消化后加入新鲜的DMEM/F12完全培养基使其重悬制成的干细胞悬液。
检测方法：因为在目标基因座上插入的T2A-EGFP序列表达绿色荧光蛋白，使用流式细胞仪检测并统计能发出绿色荧光的细胞数量以及细胞总量。将能发出绿色荧光的细胞数量除以细胞总量可以得到总的编辑效率。
实验结果：实施例4-1编辑效率为79.4％，实施例4-2编辑效率为75.3％，对比例1编辑效率为9.5％。
2、PCR检测等位基因插入效率
检测样品：96孔板培养的单克隆细胞株(A板和B板)
检测方法：在同源臂外设计引物。引物由生工生物工程(上海)股份有限公司合成。
实施例4-1、对比例4-1使用SPP1-FW、SPP1-REV。实施例4-2使用COL1A1-FW、COL1A1-REV。
将96孔板中的克隆所获得的基因组进行PCR扩增。将PCR产物进行电泳并分析。实施例4-1、对比例4-1只有1144bp左右条带的为非编辑细胞，只有1921bp的为双等位基因编辑细胞，既有1144bp左右条带也有1921bp的为单等位基因编辑细胞。分别标注并统计这三类编辑类型的比例。实施例4-2中只有1084bp左右条带的为非编辑细胞，只有1861bp的为双等位基因编辑细胞，既有1084bp左右条带也有1861bp的为单等位基因编辑细胞。
实验结果：实施例4-1编辑效率：等位基因双编辑62.3％、等位基因单编辑18.1％，对比例4-1编辑效率：等位基因双编辑0.5％、等位基因单编辑0.8％；实施例4-2编辑效率：等位基因双编辑68.5％、等位基因单编辑15.5％；可见电转与病毒转染的时间间隔对基因敲入的效率影响较大，在优选在电转结束后5-20分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行8-24小时的转染的情况下，基因敲入的效率最高。
3、所构建细胞模型在体外诱导下所致的细胞骨分化检测
将实施例4-1得到的进行了EGFP敲入的脂肪间充质干细胞铺种于6孔板，第2天更换为成骨诱导液(10mmol/Lβ-甘油磷酸(购于Sigma-Aldrich，货号：G9422)、1×10 -7mol/L地塞米松(购 于Sigma-Aldrich，货号：D4902)、50μg/mL抗坏血酸(购于Sigma-Aldrich，货号：PHR1008)，每孔总体积为2mL。每3天更换1次成骨诱导液。在诱导第0天、9天、12天、15天、18天、27天依次收集处理后的细胞。将收集的细胞固定后进行免疫细胞化学染色(一抗为Anti-Osteopontin，购于艾博抗(上海)贸易有限公司,货号：ab69498，对应的荧光二抗为Goat anti-Mouse IgG(H+L)Highly Cross-Adsorbed Secondary Antibody,Alexa Fluor Plus 555，购于ThermoFisher Scientific,Inc.，货号：A-21424)。将染色后的细胞分别使用激光共聚焦显微镜观察绿色荧光通道和红色荧光通道，最后将图层merge(重合)后进行比较。结果如表4-2所示。
表4-2体外诱导下细胞骨分化状态检测
Figure PCTCN2018119852-appb-000009
上述表4-2结果显示，实施例4-1中所建立的模型可在不致细胞死亡的情况下，实时检测诱导下的细胞骨分化。SPP1基因的表达是骨细胞分化成熟的标志之一。常规用免疫化学的方法检测SPP1基因的表达，实施例4-1中构建的EGFP敲入的脂肪间充质干细胞在成骨分化过程中实时绿色荧光检测的结果和细胞免疫化学检测的结果一致。
实施例4-2操作同实施例4-1，相应的只需要替换一抗为：Anti-Collagen I，购于艾博抗(上海)贸易有限公司，货号：ab90395。结果如表4-3所示。
表4-3体外诱导下细胞骨分化状态检测
Figure PCTCN2018119852-appb-000010
上述表4-3结果显示，实施例4-2中所建立的模型可在不致细胞死亡的情况下，实时检测诱导下的细胞骨分化。COL1A1基因的表达是骨细胞分化成熟的标志之一，常规用免疫化学的方法检测COL1A1基因的表达，实施例4-2中构建的EGFP敲入的脂肪间充质干细胞在成骨分化过程中实时绿色荧光检测的结果和细胞免疫化学检测的结果一致。
实施例5-1，用于详细说明本公开的第五种优选的实施方式。
构建在胎盘间充质干细胞的血清白蛋白基因(ALB)中插入EGFP报告基因的细胞模型。采用ALB基因原位基因启动子，在终止密码子前插入P2A-EGFP。
1、sgRNA设计以及合成
(1)通过在线sgRNA设计工具(http://crispr.mit.edu/)针对ALB基因第十四号外显子上设计靶向识别的ALB sgRNA。ALB sgRNA:5’-aaugugauguuauaagccuaguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu--3’(SEQ ID NO.98)。其中1-20位的序列为识别基序，其余的序列为tracrRNA。
(2)该sgRNA由Integrated Dna Technologies.USA公司合成并在该sgRNA序列的5’端和3’端末尾的三个碱基的二号位和三号位上分别添加O-Me、phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)基因组扩增识别靶序列片段(2000bp)，引物由生工生物工程(上海)股份有限公司合成。
ALB FW：5‘-gagtctatttgtagaaaatg-3’(SEQ ID NO.104)，
ALB REV：5‘-ctctactgaagcgactggag-3’(SEQ ID NO.105)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2μL配置成20μL反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为人胎盘间充质干细胞，根据AAV组织亲和性对照表，优选血清型为AAV-9的包装系统。
(2)AAV-9的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。
左同源臂序列为SEQ ID NO.99，其中第490-492位碱基为经过突变的PAM位点。
右同源臂序列为SEQ ID NO.100。
插入片段为P2A和EGFP，共774bp，序列如SEQ ID NO.106所示。使用无缝克隆(
Figure PCTCN2018119852-appb-000011
高保真DNA组装试剂盒，购于NEB(北京)有限公司，货号：E2621S)的方法将Cassette插入到pAAV载体上，pAAV载体序列为SEQ ID NO.107。
4、AAV病毒包装及纯化
(1)在病毒包装前一天将HEK293T细胞按照每皿5×10 6的数量种植到直径10cm的含有10mL完全培养基(DMEM+10％FBS+1％P/S双抗)(DMEM培养基，购于ThermoFisher Scientific,Inc.，货号：C11995500BT；FBS，购于ThermoFisher Scientific,Inc.，货号：sv30087.02；P/S双抗，购于ThermoFisher Scientific,Inc.，货号：SV30010)的CORNING培养皿中，共种植30皿。在37度、5％CO 2的细胞培养箱中培养24小时。
(2)病毒包装当天检查HEK293T细胞汇合度是否达到80％。每皿按照以下步骤单独配置转染体系：将10μg的pAAV-Cassette，10μg的pHelper，10μg的pAAV9-RC混合后使用无血清的DMEM调整体积到910μL后经漩涡振荡器混匀，加入90μL的PEI(购于Polysciences Asia Pacific,Inc.，货号：23966-2)后再次使用漩涡振荡器混匀，静置15分钟。将CORNING培养皿中的完全培养基替换成9mL无血清培养基，再将之前经过静置的DNA-PEI复合物均匀滴加到培养皿中，轻柔晃匀后置于37度、5％CO 2的细胞培养箱中培养6小时。转染6h后将CORNING培养皿中的无血清培养基替换成完全培养基，于37度、5％CO 2的细胞培养箱中继续培养。
(3)在转染60小时之后，分别收获上清和细胞。
将收集得到的上清经4000rpm 4度离心10分钟后弃去杂质。将去除杂质的上清加入Amicon Ultra-15超离柱中(购于默克化工技术(上海)有限公司，货号：UFC905096)，经过若干次4000rpm、4度离心30分钟将体积浓缩至10到15mL。将用细胞刮刀刮下的HEK293T细胞用适量培养基吹匀并转移至50mL离心管中，经1500rpm、4度离心10min后弃上清，所有沉淀总共加3mL细胞裂解缓冲液(150mM NaCl,20mM tris pH8.0)使其重悬。将重悬细胞在-80℃酒精浴和37℃水浴中反复冻融三次。将浓缩的上清和冻融的细胞悬液混匀，添加1M MgCl 2至终浓度为1mM。添加Benzonase(购于默克化工技术(上海)有限公司，货号：70746-1kU)至终浓度为25U/mL，混匀后37℃反应40min。取出50mL离心管，4℃，4000rpm离心20min，取上清。
(4)采用碘克沙醇密度梯度离心法纯化病毒。
(5)利用qPCR对AAV9进行滴度检测。根据EGFP序列设计引物，使qPCR产物的长度约200bp。qPCR引物由生工生物工程(上海)股份有限公司合成。
AAVGFPF:5’-tcagcttcaggcaccaccac-3’(SEQ ID NO.108)。
AAVGFPR:5’-tgaacttgtggccgtttacgtcg-3’(SEQ ID NO.109)。
5、RNP复合物组装及待编辑细胞的预处理
(1)将SpCas9(终浓度300μg/ml)和ALB sgRNA(终浓度175μg/ml)按照1:3的摩尔比进行混合在室温孵育10min，从而形成SpCas9/sgRNA RNP复合物。
(2)胎盘间充质干细胞汇合率达到80％后制成干细胞悬液，调整细胞密度到5×10 7/mL。
(3)将10μL经过室温孵育的SpCas9/sgRNA SNP复合物(以sgRNA的量计，所述RNP复合物的含量为7.5μmol)和10μL经过计数并使用Opti-MEM重悬的胎盘间充质干细胞悬液(其中细胞数量为5×10 5个)相混合，将20μL混合液转移到16孔电转板条中。使用Lonza 4D核转染系统选择CB150模式(电场强度为150V/cm，单次脉冲时间为10ms，相邻两次脉冲之间的时间间隔为20s，总脉冲次数为5次)进行电转。电转完成后立刻将细胞转移到含DMEM/F12完全培养基的24孔板中于37度、5％CO 2的细胞培养箱中继续培养。
(4)电转完第5到20分钟内开始按照1×10 5的MOI值轻柔得滴加AAV9，并于电转完20分钟内滴加完毕。
(5)电转完24小时后将旧培养基移除，更换为DMEM/F12完全培养基。
6、单克隆细胞株的培养：
(1)电转完第48小时使用PBS漂洗一次培养皿。加入胰酶使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去胰酶。即刻加入新鲜的DMEM/F12完全培养基，用1mL枪扇形吹打培养皿/瓶底，使皿底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，保证细胞间没有粘连，制成干细胞悬液。使用血球计数板对细胞密度进行计数。按每个10cm培养皿15000个细胞的比例把细胞种植到含DMEM/F12完全培养基的10cm CORNING培养皿中于37度、5％CO 2的细胞培养箱中继续培养。
(2)每24小时进行观察并移除旧的培养基，更换为新的DMEM/F12完全培养基。72小时后可以在镜下观察到克隆的形成。
(3)电转后12到14天在十倍的物镜下可以看到克隆的大小已经相当于一个硬币。不能让克隆继续变大或者相交。在96孔板上的每个孔中加入120μL DMEM/F12完全培养基，标记板O。在超净台中通过显微镜进行观察，将P200移液器调节至45μL使用带有滤芯的枪头刮碎克隆，用移液器收集细胞并转移到96孔板的小孔中。
(4)克隆挑取之后每24小时后将旧的培养基移除，更换为新的DMEM/F12完全培养基，四天后细胞汇合率达到80％。
(5)在两块96孔板上的每个孔中加入133μL DMEM/F12完全培养基，分别标记为板A、板B。将板O的每孔使用150μL的PBS漂洗。每孔中加入35μL的胰酶，消化5到10分钟后每孔中添加165μL的DMEM/F12完全培养基。从孔中分别移取66μL的细胞悬液转移到板A和板B的对应小孔中。板A用于基因组提取，板B备用。在板O的每个孔中直接添加165μL的细胞冻存液后至于深低温冰箱存储。
实施例5-2
构建在胎盘间充质干细胞，甲种胎儿球蛋白基因(AFP)中插入EGFP报告基因的细胞模型。采用AFP基因原位基因启动子，在终止密码子前插入P2A-EGFP。因为AFP的两个剪辑变体变体拥有相同的CTD所以在本实施例中同时对AFP的两种剪辑变体均进行了标记。。
通过在线sgRNA设计工具(http://crispr.mit.edu/)在COL1A1基因51号外显子靠近终止密码子(TAA)上游100bp以内的序列上设计靶向识别的sgRNA。
AFP sgRNA:5’-gguuguagaccccaaaaguaguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacu ugaaaaaguggcaccgagucggugcuuuuuuu-3’。(SEQ ID NO.101)。
其中1-20位的序列为识别基序，其余的序列为tracrRNA。
该sgRNA由Integrated Dna Technologies.USA公司合成并在序列的5’端和3’端末尾的三个碱基的二号位和三号位上分别添加-2-O-Me、-3-phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)从基因组扩增识别靶序列片段(2000bp)，引物由生工生物工程(上海)股份有限公司合成。
AFP FW：5‘-gagtctatttgtagaaaatg-3’，(SEQ ID NO.110)，
AFP REV：5‘-ctctactgaagcgactggag-3’，(SEQ ID NO.111)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2μl配置成20μl反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为胎盘间充质干细胞株，根据AAV组织亲和性对照表，优选血清型为AAV9的包装系统。
(2)AAV9的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。左同源臂序列为SEQ ID NO.102，其中第493-495位碱基为经过突变的pam位点)，右同源臂序列为SEQ ID NO.103。插入片段为P2A和EGFP，共774bp，序列如SEQ ID NO.108所示。使用无缝克隆(
Figure PCTCN2018119852-appb-000012
高保真DNA 组装试剂盒，购于NEB(北京)有限公司)的方法将Cassette插入到pAAV载体上，载体序列为SEQ ID NO.132。为了避免SpCas9/sgRNA RNP复合物对重组位点的二次切割从而影响基因编辑的效率，通过PrimerStar高保真DNA聚合酶(购于宝生物工程(大连)有限公司，货号：R044A)利用PCR刻环的方法在PAM位点引入了点突变(CGG突变为CTG)，从而避免了SpCas9/sgRNA RNP复合物对识别位点的切割。点突变引物由生工生物工程(上海)股份有限公司合成。
AFP-PAM-mut-FW:5’-aaactcgtgctgctttaggagtttaaattactt-3’(SEQ ID NO.112)。
AFP-PAM-mut-Rev:5’-aagtaatttaaactcctaaagcagcacgagttt-3’(SEQ ID NO.113)。
4、AAV病毒包装及纯化
同实施例5-1
5、RNP复合物组装及待编辑细胞的预处理
同实施例5-1
6、Lonza 4D系统电转及AAV感染
同实施例5-1
7、单克隆细胞株的培养
同实施例5-1
对比例5-1
按照实施例5-1的方法进行，不同的是电转完第35min开始按照1×10 5的MOI轻柔地滴加AAV9，并于电转完50分钟内滴加完毕。
测试实施例5-1
1、荧光检测等位基因插入效率检测
检测样品：实施例5-1、5-2以及对比例5-1经过电转和AAV感染后第四天经过胰酶消化后加入新鲜的DMEM/F12完全培养基使其重悬制成的干细胞悬液。
检测方法：因为在目标基因座上插入的P2A-EGFP序列表达绿色荧光蛋白，使用流式细胞仪检测并统计能发出绿色荧光的细胞数量以及细胞总量。将能发出绿色荧光的细胞数量除以细胞总量可以的到总的编辑效率。
实验结果：实施例5-1编辑效率为78.1％，实施例5-2编辑效率为76.6％，对比例5-1编辑效率为1.2％。
2、PCR检测等位基因插入效率
检测样品：96孔板培养的单克隆细胞株(A板和B板)
检测方法：在同源臂外设计引物。引物由生工生物工程(上海)股份有限公司合成。
实施例5-1、对比例5-1使用ALB-FW、ALB-REV。实施例5-2使用AFP-FW、AFP-REV。
将96孔板中的克隆所获得的基因组进行PCR扩增。将PCR产物进行电泳并分析。实施例5-1、对比例5-1只有2000bp左右条带的为非编辑细胞，只有2800bp的为双等位基因编辑细胞，既有2000bp左右条带也有2800bp的为单等位基因编辑细胞。分别标注并统计这三类编辑类型的比例。实施例5-2中只有2000bp左右条带的为非编辑细胞，只有2800bp的为双等位基因编辑细胞，既有2000bp左右条带也有2800bp的为单等位基因编辑细胞。分别标注并统计这三类编辑类型的比例。
实验结果：实施例5-1编辑效率：等位基因双编辑30.2％、等位基因单编辑55.3％，对比例5-1编辑效率：等位基因双编辑0.9％、等位基因单编辑1.5％；实施例5-2编辑效率：等位基因双编辑23.4％、等位基因单编辑58.8％；可见电转与病毒转染的时间间隔对基因敲入的效率影响较大，在优选在电转结束后5-20分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行8-24小时的转染的情况下，基因敲入的效率最高。
3、所构建细胞模型在体外诱导下向成熟肝样细胞分化检测
参照Taléns-Visconti R在文章Human mesenchymal stem cells from adipose tissue:Differentiation into hepatic lineage.中的方法对实施例5-1中在ALB敲入EGFP的胎盘间充质干细胞以及实施例2中在AFP敲入EGFP的胎盘间充质干细胞上进行诱导使其分化为成熟的肝样细胞。在使用第四诱导培养基诱导细胞分化成熟的过程中(总诱导第13、15、17、19、21天)时将细胞固定后进行免疫细胞化学染色(ALB的一抗为Anti-Albumin抗体购艾博抗(上海)贸易有限公司，货号：ab106582，对应的荧光二抗为Goat anti-Chicken IgY(H+L)Secondary Antibody,Alexa Fluor 555购于ThermoFisher Scientific,Inc.，货号：A-21424；AFP的一抗为：Anti-alpha 1Fetoprotein抗体[AFP-01]购艾博抗(上海)贸易有限公司，货号：ab3980，对应的荧光二抗为Goat anti-Mouse IgG(H+L)Highly Cross-Adsorbed Secondary Antibody,Alexa Fluor Plus 555购于ThermoFisher Scientific,Inc.，货号：A-21424)。将两组细胞分别使用荧光显微镜观察绿色荧光通道和红色荧光通道，将图层merge(重合)之后统计绿色荧光和红色荧光观察的重合率。
表5-1实施例5-1中细胞肝样分化状态检测
Figure PCTCN2018119852-appb-000013
表5-2实施例5-2中细胞肝样分化状态检测
Figure PCTCN2018119852-appb-000014
从表5-1和表5-2的统计数据上可以看出，EGFP所指示的细胞与实施例5-1中ALB以及实施 例5-2中AFP所指示的细胞相一致。也就是说在通过实施例5-1和实施例5-2所建立的EGFP标记的胎盘间充质干细胞可可以实时检测其肝样分化的程度可以实时检测其肝样分化的程度可以实时检测其肝样分化的程度。
实施例6-1，用于说明本公开的第六种优选实施方式。
构建在1016细胞株(购自美国哈佛大学干细胞库)的安全位点(RNA45SN4)中插入GPX7基因表达Cassette的细胞模型。
1、sgRNA设计以及合成
(1)通过在线sgRNA设计工具(http://crispr.mit.edu/)在基因插入安全岛(RNA45SN4)上设计靶向识别的RNA45SN4sgRNA，优选结果如下：5’-acgccgcggcggccgucgggguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuuuuu-3’(SEQ ID NO.114)。其中1-20位的序列为识别基序，其余的序列为tracrRNA。
(2)该sgRNA由Integrated DNA Technologies US公司合成并在该sgRNA序列的5’端和3’端末尾分的三个碱基的二号位和三号位上别添加O-Me、phosphorothioate的修饰。
2、体外法检测sgRNA的活性
(1)从基因组扩增识别靶序列片段(2000bp)，引物由生工生物工程(上海)股份有限公司合成。
GPx7FW：5’-gaggaattcccagtaagtgc-3’(SEQ ID NO.118)，
GPx7REV：5’-gcgctcccccgaccctctct-3’(SEQ ID NO.119)。
(2)将PCR扩增产物200ng，sgRNA 100ng，SpCas9 200ng(购于Sigma-Aldrich，货号：TGEN-CP-500UG)，10×缓冲液2μL配置成20μL反应体系。反应体系在37度保温一小时，在70度保温一小时。
(3)使用TAE配置1％的BioWest琼脂糖胶，在90V的电压下电泳30min，使用凝胶成像仪观察结果。
3、AAV包装系统选择及质粒构建
(1)待编辑细胞为1016细胞株，根据AAV组织亲和性对照表，优选血清型为AAV-8的包装系统。
(2)AAV-8的总包装容量为4.7Kb。在AAV包装系统的装载载体上所插入的片段应包含左同源臂(500bp左右)，插入片段以及右同源臂(500bp左右)。
左同源臂序列为SEQ ID NO.115。
右同源臂序列为SEQ ID NO.116，其中第4-7位碱基为经过突变的PAM位点。
插入片段为一个完整的Cassette包括CMV增加子、CMV启动子、GPX7CDS和bGH poly A，共1296bp，序列如SEQ ID NO.4所示，其中第1-507位为启动子，第508-1071位为EGFP，第1072-1296位为bGH polyA。.使用NheI和BsmI限制性内切酶(购于NEB(北京)有限公司)将Cassette插入到pAAV载体上，载体序列为SEQ ID NO.7。为了避免SpCas9/sgRNA RNP复合物对重组位点的二次切割从而影响基因编辑的效率，通过PrimerStar高保真DNA聚合酶(购于宝生物工程(大连)有限公司，货号：R044A)利用PCR刻环的方法在PAM位点引入了点突变(TGG突变为TAG)，从而避免了了SpCas9/sgRNA RNP复合物对识别位点的切割。点突变引物由生工生物工程(上海)股份有限公司合成。
GPx7-PAM-mut-FW:5’-gcggcggccgtcgggtaggggctttacccggcg-3’(SEQ ID NO.121)；
GPx7-PAM-mut-REV:5’-cgccgggtaaagcccctacccgacggccgccgc-3’(SEQ ID NO.122)。
4、AAV病毒包装及纯化
(1)在病毒包装前一天将HEK293T细胞按照每皿5×10 6的数量种植到直径10cm的含有10mL 完全培养基(DMEM+10％FBS+1％P/S双抗)(DMEM培养基，购于ThermoFisher Scientific,Inc.，货号：C11995500BT；FBS，购于ThermoFisher Scientific,Inc.，货号：sv30087.02；P/S双抗，购于ThermoFisher Scientific,Inc.，货号：SV30010)的CORNING培养皿中，共种植30皿。在37度、5％CO 2的细胞培养箱中培养24小时。
(2)病毒包装当天检查HEK293T细胞汇合度是否达到80％。每皿按照以下步骤单独配置转染体系：将10μg的pAAV-Cassette，10μg的pHelper，10μg的pAAV9-RC混合后使用无血清的DMEM调整体积到910μL后经漩涡振荡器混匀，加入90μL的PEI(购于Polysciences Asia Pacific,Inc.，货号：23966-2)后再次使用漩涡振荡器混匀，静置15分钟。将CORNING培养皿中的完全培养基替换成9mL无血清培养基，再将之前经过静置的DNA-PEI复合物均匀滴加到培养皿中，轻柔晃匀后置于37度、5％CO 2的细胞培养箱中培养6小时。转染6h后将CORNING培养皿中的无血清培养基替换成完全培养基，于37度、5％CO 2的细胞培养箱中继续培养。
(3)在转染60小时之后，分别收获上清和细胞。
将收集得到的上清经4000rpm 4度离心10分钟后弃去杂质。将去除杂质的上清加入Amicon Ultra-15超离柱中(购于默克化工技术(上海)有限公司，货号：UFC 905096)，经过若干次4000rpm、4度离心30分钟将体积浓缩至10到15mL。将用细胞刮刀刮下的HEK293T细胞用适量培养基吹匀并转移至50mL离心管中，经1500rpm、4度离心10min后弃上清，所有沉淀总共加3mL细胞裂解缓冲液(150mM NaCl,20mM tris pH8.0)使其重悬。将重悬细胞在-80℃酒精浴和37℃水浴中反复冻融三次。将浓缩的上清和冻融的细胞悬液混匀，添加1M MgCl 2至终浓度为1mM。添加Benzonase(购于默克化工技术(上海)有限公司，货号：70746-1kU)至终浓度为25U/mL，混匀后37℃反应40min。取出50mL离心管，4℃，4000rpm离心20min，取上清。
(4)采用碘克沙醇密度梯度离心法纯化病毒。
(5)利用qPCR对AAV8进行滴度检测。根据EGFP序列设计引物，使qPCR产物的长度约200bp。qPCR引物由生工生物工程(上海)股份有限公司合成。
AAV-GPx7-F:5’-actggtgtcgctggagaagt-3’(SEQ ID NO.123)。
AAV-GPx7-R:5’-caatctccttgttgctgtcag-3’(SEQ ID NO.124)。
5、RNP复合物组装及待编辑细胞的预处理
(1)将SpCas9(终浓度1μmol/μL)和RNA45SN4sgRNA(终浓度1μmol/μL)按照1:3的摩尔比进行混合在室温孵育10min，从而形成SpCas9/sgRNA RNP复合物。
(2)1016细胞株汇合率达到80％后制成干细胞悬液，调整细胞密度到5×10 7/mL。
(3)将10μL经过室温孵育的SpCas9/sgRNA SNP复合物(以sgRNA的量计，所述RNP复合物的含量为7.5μmol)和10μL经过计数并使用Opti-MEM重悬的1016细胞悬液(其中细胞数量为5×10 5个)相混合，将20μL混合液转移到16孔电转板条中。使用Lonza 4D核转染系统选择CB150模式(电场强度为150V/cm，单次脉冲时间为10ms，相邻两次脉冲之间的时间间隔为20s，总脉冲次数为5次)进行电转。电转完成后立刻将细胞转移到经过Geltrex包被(购于ThermoFisher Scientific,Inc.，货号：A1413202)并且添加了500μL mTeSR和10μM Y-27632(购于Stem Cell Technologies,Inc.，货号：72304)的24孔板中于37度、5％CO 2的细胞培养箱中继续培养。
(4)电转完第5到20分钟内开始按照1×10 5的MOI值轻柔地滴加AAV-DJ，并于电转完20分钟内滴加完毕。
(5)电转完24小时后将旧培养基移除，更换为新的mTeSR培养基(含有10μM的Y-27632)
6、单克隆细胞株的培养：
(1)电转完第48小时使用PBS漂洗一次培养皿。加入Accutase消化酶使之完全覆盖皿底。37℃孵育3-5分钟后停止消化，吸去Accutase消化酶。即刻加入新鲜的mTeSR培养基，用1mL枪扇形吹 打培养皿/瓶底，使皿底贴附的干细胞集落脱落，轻柔缓慢吹吸混匀，保证细胞间没有粘连，制成干细胞悬液。使用血球计数板对细胞密度进行计数。按每个10cm培养皿15000个细胞的比例把细胞种植到经过Geltrex包被并且添加了10mL mTeSR和10μM Y-27632的10cm CORNING培养皿中于37度、5％CO 2的细胞培养箱中继续培养。
(2)每24小时进行观察并移除旧的培养基，更换为新的mTeSR培养基(含有10μM的Y-27632)。72小时后可以在镜下观察到克隆的形成。
(3)电转后12到14天在十倍的物镜下可以看到克隆的大小已经相当于一个硬币。不能让克隆继续变大或者相交。在经过Geltrex包被的96孔板上的每个孔中加入120μL mTeSR(含有10μM Y-27632)，标记板O。在超净台中通过显微镜进行观察，将P200移液器调节至45μL使用带有滤芯的枪头刮碎克隆，用移液器收集细胞并转移到96孔板的小孔中。
(4)克隆挑取之后每24小时后将旧的培养基移除，更换为新的mTeSR培养基(含有10μM的Y-27632)四天后细胞汇合率达到80％。
(5)在两块经过Geltrex包被的96孔板上的每个孔中加入133μL mTeSR(含有10μM Y-27632)，分别标记为板A、板B。将板O的每孔使用150μL的PBS漂洗。每孔中加入35μL的Accutase消化酶，消化5到10分钟后每孔中添加165μL的mTeSR(含有10μM Y-27632)。从孔中分别移取66μL的细胞悬液转移到板A和板B的对应小孔中。板A用于基因组提取，板B备用。在板O的每个孔中直接添加165μL的mFreSR(购于Stem Cell Technologies,Inc.)后至于深低温冰箱存储。
7、基因编辑效率检测(等位基因插入检测)
(1)在同源臂外设计引物(非整合位点PCR产物约2000bp左右，整合位点PCR产物3400bp)。引物由生工生物工程(上海)股份有限公司合成。
GPx7FW：5’-gaggaattcccagtaagtgc-3’(SEQ ID NO.118)，
GPx7REV：5’-gcgctcccccgaccctctct-3’(SEQ ID NO.119)。
(2)从96孔板中挑取克隆，抽提基因组后进行PCR扩增。
(3)将PCR产物进行电泳并分析。只有2000bp左右条带的为非编辑细胞，既有2000bp左右条带也有3400bp的为编辑细胞。分别标注并统计这两类编辑类型的比例结果如表6-1所示。
表6-1
  非编辑细胞比例 基因编辑细胞
实施例6-1 25％ 75％
8、多能干细胞定向诱导分化成为MSC
将GPx7插入的1016细胞株进行拟胚胎(EB)分化
准备300-500个细胞，大小均一的GPx7插入1016克隆，用室温PBS清洗一次，用Accutase消化酶37度消化3-5分钟。待1016克隆形成球体后，用CDF12培养基重悬后，加到低粘附培养板中(货号3471，购于CORNING公司)。37度、5％CO 2的细胞培养箱中继续培养1到3天后即形成拟胚体。
将获得的拟胚体接种于基质胶(geltrex，购于ThermoFisher Scientific,Inc.，)包被的六孔板中进行培养，继续培养2周至纤维状细胞出现。再经过一次传代后，利用流式细胞术分选其中CD73、CD90、CD105均为阳性的细胞群体，此即为GPx7增强型MSC。
对比例6-1：脐带间充质干细胞
对比例6-1培养方法参照《改良的人脐带间充质干细胞培养方法》中所述方法。
测试实施例：
实施例6-1得到的GPx7增强型MSC体外传代后的衰老状态检测(相对于对比例1)
细胞衰老β-半乳糖苷酶染色是一种基于衰老时SA-β-Gal活性水平的上调而对衰老细胞或组织进行染色检测的方法。在体外传代10、15代的情况下，分别对从实施例6-1中获得的GPx7增强型MSC和对比例6-1的脐带间充质干细胞进行SA-β-Gal染色，并在普通的光学显微镜下观察到细胞或组织的衰老情况，并对两组细胞中的SA-β-Gal阳性细胞染色比率进行定量统计分析。
染色步骤：用PBS洗涤一次供体细胞，再加入染色固定液(2％甲醛+0.2％戊二醛)，室温固定5分钟。弃去固定液，用PBS洗涤1次，每孔加入1ml染色工作液。以X-Gal作为底物，在衰老特异性的β半乳糖苷酶会生成深蓝色产物。实验结果：衰老细胞阳性比率：对比例6-1>实施例6-1，结果如表6-2所示。
表6-2
Figure PCTCN2018119852-appb-000015
将实施例6-1中获得的GPx7增强型MSC和对比例6-1的脐带间充质干细胞在体外传代10到15代后分别对衰老高表达基因p16、p21、GATA4的蛋白表达情况进行蛋白质免疫印迹检测。一抗分别为p16(购于BD，货号：550834)、p21(购于Cell Signaling，货号：2947)，GATA4(购于Santa Cruz，货号：SC-1237)、Actin(购于Sigma-Aldrich，货号：A1978)，使用的HRP标记的二抗购于中杉金桥分别为羊抗小鼠，货号：ZDR5307，羊抗兔，货号：ZDR5306，兔抗羊，货号：ZDR5308。
实验结果：p16、p21、GATA4在GPx7增强型MSC中的表达丰度低于其在对比例1的脐带间充质干细胞中的表达丰度。相应蛋白表达丰度＝相应蛋白灰度值/actin条带灰度值，结果如表3所示。
表6-3
Figure PCTCN2018119852-appb-000016
3、实施例6-1得到的GPx7增强型MSC和对比例6-1的脐带间充质干细胞对血糖持续调节作用的检测
注射到STZ造模大鼠体内，STZ大鼠造模参照《照脂肪间充质干细胞及其外泌体对炎症状态脂肪细胞及2型糖尿病大鼠的作用及机制》中所述方法。将实施例1得到的GPx7增强型MSC细胞和对比例1的脐带间充质干细胞，按照1ml浓度剂量2x10 6cell/ml的细胞量通过尾静脉注射至STZ大鼠体内，分别在注射细胞5天，10，天，15天，20天时尾静脉取血，检测空腹血糖浓度。结果如表6-4
表6-4
空腹血糖(mM) d5 d10 d15 d20
实施例6-1 4.6 6.1 7.2 10.1
对比例6-1 5.1 11.1 15.6 20.8
如表6-4所示，实施例6-1相较于对比例6-1具有长时间维持调节血糖作用的能力。
以上详细描述了本公开的优选实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。

Claims (25)

  1. 一种在干细胞中进行基因定向敲入的方法，其特征在于，该方法包括如下步骤：
    S1、将针对待敲入的靶位点的sgRNA和Cas9蛋白混合以形成RNP复合物；
    S2、将插入有模板DNA的同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；
    S3、将干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；
    S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；
    S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
  2. 根据权利要求1所述的方法，其中，所述模板DNA包括左同源臂序列、敲入片段和右同源臂序列；优选所述敲入片段包括增强子、启动子、敲入基因和polyA；所述敲入基因的长度为200-5000bp；优选所述敲入基因为报告基因，进一步优选所述报告基因为荧光蛋白报告基因。
  3. 根据权利要求1所述的方法，其中，所述待敲入的靶位点的sgRNA的序列如SEQ ID NO.1所示；所述模板DNA的序列如SEQ ID NO.6所示；所述载体的骨架序列如SEQ ID NO.7所示。
  4. 根据权利要求1所述的方法，其中，该方法为制备可实时检测间充质干细胞衰老的细胞模型的方法，且该方法包括如下步骤：
    S1、将针对衰老相关基因的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；所述衰老相关基因包括Klotho、p16、p21、p53、GATA4、SIRT1、SIRT3、SIRT6和MORF4中的至少一种；所述衰老相关基因的敲入位点为所述衰老相关基因的最末一个有义密码子和终止密码子之间；
    S2、将插入有模板DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模板DNA包括针对所述衰老相关基因的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有自剪切2A肽编码序列和荧光蛋白报告基因，以使得定向敲入后形成编码由衰老相关基因、2A肽和荧光蛋白串联而成的融合蛋白的融合基因；
    S3、将间充质干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；
    S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；
    S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
  5. 根据权利要求4所述的方法，其中，所述自剪切2A肽为T2A肽、F2A肽和P2A肽中的至少一种；所述荧光蛋白报告基因为EGFP、ECFP、EYFP、ERFP、mCherry、tdTomato和Venus中的至少一种。
  6. 根据权利要求4所述的方法，其中，所述sgRNA如SEQ ID NO.14、17、20、23、26、29、32、35或38所示；相应地，所述左同源臂序列如SEQ ID NO.15、18、21、24、27、30、33、36或39所示；相应地，所述右同源臂序列如SEQ ID NO.16、19、22、25、28、31、34、37或40所示。
  7. 根据权利要求4所述的方法，其中，该方法为可解除免疫抑制并增强肿瘤靶向性杀伤的间充 质干细胞的制备方法，该方法包括如下步骤：
    S1、将针对PD-1基因的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；
    S2、将插入有模版DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模版DNA包括针对PD-1基因的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有TRAIL基因；且能够通过TRAIL基因的定向敲入使得PD-1基因的表达沉默；
    S3、将间充质干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；
    S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；
    S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
  8. 根据权利要求7所述的方法，其中，所述PD-1基因的敲入位点位于PD-1基因的第一外显子上；所述左同源臂序列和所述右同源臂序列之间插入有CMV增强子、CMV启动子、TRAIL基因的表达框和SV40poly A；所述TRAIL基因为膜结合型TRAIL基因或分泌型TRAIL基因；优选地，所述左同源臂序列和所述右同源臂序列之间插入的序列如SEQ ID NO.56或SEQ ID NO.62所示。
  9. 根据权利要求7所述的方法，其中，所述sgRNA如SEQ ID NO.53所示；相应地，所述左同源臂序列如SEQ ID NO.54所示；相应地，所述右同源臂序列如SEQ ID NO.55所示。
  10. 根据权利要求1所述的方法，其中，该方法为可实时检测间充质干细胞骨分化能力的细胞模型的方法，且该方法包括如下步骤：
    S1、将针对骨分化相关基因的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；所述骨分化相关基因包括SPP1、COL1A1、BMP-2、Runx2、SLP1、IBSP及BGLAP中的至少一种；所述骨分化相关基因的敲入位点为所述骨分化相关基因的最末一个有义密码子和终止密码子之间；
    S2、将插入有模板DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模板DNA包括针对所述骨分化相关基因的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有自剪切2A肽编码序列和荧光蛋白报告基因，以使得定向敲入后形成编码由骨分化相关基因、2A肽和荧光蛋白串联而成的融合蛋白的融合基因；
    S3、将间充质干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；
    S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；
    S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
  11. 根据权利要求10所述的方法，其中，所述自剪切2A肽为T2A肽、F2A肽和P2A肽中的至少一种；所述荧光蛋白报告基因为EGFP、ECFP、EYFP、GFP、RFP、mCherry、tdTomato和Venus中的至少一种。
  12. 根据权利要求10所述的方法，其中，所述sgRNA如SEQ ID NO.65、68、71、74、77、80 或83所示；相应地，所述左同源臂序列如SEQ ID NO.66、69、72、75、78、81或84所示；相应地，所述右同源臂序列如SEQ ID NO.67、70、73、76、79、82或85所示。
  13. 根据权利要求1所述的方法，其中，该方法为一种制备可实时检测间充质干细胞向成熟肝样细胞分化的细胞模型的方法，且该方法包括如下步骤：
    S1、将针对肝样细胞相关基因的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；所述肝样细胞相关基因包括ALB和/或AFP；所述肝样细胞相关基因的敲入位点为所述肝样细胞相关基因的最末一个有义密码子和终止密码子之间；
    S2、将插入有模板DNA同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模板DNA包括针对所述肝样细胞相关基因的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有自剪切2A肽编码序列和荧光蛋白报告基因，以使得定向敲入后形成编码由肝样细胞相关基因、2A肽和荧光蛋白串联而成的融合蛋白的融合基因；
    S3、将间充质干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；
    S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；
    S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株。
  14. 根据权利要求13所述的方法，其中，所述自剪切2A肽为T2A肽、F2A肽和P2A肽中的至少一种；所述荧光蛋白报告基因为EGFP、ECFP、EYFP、GFP、RFP、mCherry、tdTomato和Venus中的至少一种。
  15. 根据权利要求13所述的方法，其中，所述sgRNA如SEQ ID NO.98或101所示；相应地，所述左同源臂序列如SEQ ID NO.99或102所示；相应地，所述右同源臂序列如SEQ ID NO.100或103所示。
  16. 根据权利要求1所述的方法，其中，该方法为制备抵抗细胞衰老及延长血糖调节作用时程的间充质干细胞的方法，且该方法包括如下步骤：
    S1、将针对基因插入安全岛的敲入位点的sgRNA和Cas9蛋白混合以形成RNP复合物；
    S2、将插入有模板DNA的同源重组载体包装到AAV病毒中，形成待转染AAV病毒颗粒；所述模板DNA包括针对基因插入安全岛的敲入位点的左同源臂序列和右同源臂序列，所述左同源臂序列和所述右同源臂序列之间插入有GPx7基因；
    S3、将iPSC细胞或胚胎干细胞的悬液与所述RNP复合物的悬液混合并进行电转，得到电转后的培养物；
    S4、在电转结束后1-30分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行4-30小时的转染，得到转染后的培养物；
    S5、将所述转染后的培养物极限稀释后进行单克隆化培养，并通过PCR及测序筛选出进行了基因定向敲入的单克隆细胞株；
    S6、将所述进行了基因定向敲入的单克隆细胞株扩增并定向诱导分化成为间充质干细胞。
  17. 根据权利要求16所述的方法，其中，所述iPSC细胞为1016细胞株，所述AAV病毒的血清型为AAV-8病毒、AAV-6病毒、AAV-1病毒或AAV9病毒；
    所述基因插入安全岛为RNA45SN4、rDNA 28S、rDNA 18S、rDNA 45S、CLYBL或AAVS1；所述左同源臂序列和所述右同源臂序列之间插入有CMV增强子、CMV启动子、GPx7基因的表达框和bGH poly A；所述GPx7基因为GPx7基因或分泌型GPx7基因；优选地，所述左同源臂序列和所述右同源臂序列之间插入的序列如SEQ ID NO.117所示；
    所述sgRNA如SEQ ID NO.114所示；相应地，所述左同源臂序列如SEQ ID NO.115所示；相应地，所述右同源臂序列如SEQ ID NO.116所示；
    扩增并定向诱导分化的条件包括：将所述进行了基因定向敲入的单克隆细胞株在低粘附培养板中继续培养18-100小时后形成拟胚体；然后将所述拟胚体接种于基质胶上继续培养10-20天后用流式细胞术分选其中CD73、CD90和CD105均为阳性的细胞群体。
  18. 根据权利要求1所述的方法，其中，所述sgRNA带有化学修饰基团，优选所述化学修饰基团为甲基化学修饰基团或磷硫酰化学修饰基团；针对待编辑的靶位点的sgRNA和Cas9蛋白的用量摩尔比为1:1至1:5。
  19. 根据权利要求1所述的方法，其中，将针对待编辑的靶位点的sgRNA和Cas9蛋白混合的时间为5-20分钟，温度为10-40℃。
  20. 根据权利要求1所述的方法，其中，步骤S3中，所述干细胞的悬液与所述RNP复合物混合时，相对于每10 6个所述干细胞，以sgRNA的量计，所述RNP复合物的用量为10-50μmol，所述干细胞的悬液中，细胞浓度为(1-5)×10 7个/mL；以sgRNA的量计，所述RNP复合物的终浓度为0.1-1.5μmol/μL。
  21. 根据权利要求1所述的方法，其中，步骤S3中，电转的条件包括：电场强度为50-250V/cm，单次脉冲时间为2-15ms，相邻两次脉冲之间的时间间隔为10-60s，总脉冲次数为2-10次。
  22. 根据权利要求1所述的方法，其中，步骤S4中，在电转结束后5-20分钟内，向所述电转后的物料中加入待转染的AAV病毒颗粒的悬液以进行8-24小时的转染，得到转染后的培养物。
  23. 根据权利要求1所述的方法，其中，步骤S4中，待转染的AAV病毒颗粒的悬液的加入量使得待转染的AAV病毒颗粒的MOI值为10 4-10 6。
  24. 权利要求16或17所述的方法制备得到的间充质干细胞。
  25. 权利要求24所述的间充质干细胞的用途，其特征在于，所述用途为如下任意一种：
    制备改善胰岛素抵抗和/或慢性炎症的产品；
    制备细胞移植治疗的产品；
    制备改善β细胞功能的治疗性产品；
    制备再生和/或修复损伤血管的产品。
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CN201810784680.9A CN109082442B (zh) 2018-07-17 2018-07-17 一种可解除免疫抑制并增强肿瘤靶向性杀伤的间充质干细胞的制备方法
CN201810785793.0A CN109097333B (zh) 2018-07-17 2018-07-17 抵抗细胞衰老及延长血糖调节作用时程的间充质干细胞及其制备方法和用途
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