WO2019028686A1 - 基因敲除方法 - Google Patents

基因敲除方法 Download PDF

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WO2019028686A1
WO2019028686A1 PCT/CN2017/096510 CN2017096510W WO2019028686A1 WO 2019028686 A1 WO2019028686 A1 WO 2019028686A1 CN 2017096510 W CN2017096510 W CN 2017096510W WO 2019028686 A1 WO2019028686 A1 WO 2019028686A1
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cell
target sequence
universal
sequence
donor dna
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PCT/CN2017/096510
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French (fr)
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魏文胜
陈一欧
周悦欣
张鸿敏
袁鹏飞
刘源
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北京大学
博雅缉因（北京）生物科技有限公司
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Application filed by 北京大学, 博雅缉因（北京）生物科技有限公司 filed Critical 北京大学
Priority to JP2020529784A priority Critical patent/JP7109009B2/ja
Priority to CN201780093414.5A priority patent/CN111278983A/zh
Priority to PCT/CN2017/096510 priority patent/WO2019028686A1/zh
Priority to US16/637,591 priority patent/US11624077B2/en
Priority to EP17920723.8A priority patent/EP3666898A4/en
Publication of WO2019028686A1 publication Critical patent/WO2019028686A1/zh

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    • 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
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    • 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
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12N9/22—Ribonucleases RNAses, DNAses
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3517—Marker; Tag

Definitions

  • the present invention relates to gene editing techniques, and in particular to gene knockout methods.
  • ZFN zinc finger nuclease
  • TALENs transcriptional activator-like effector nuclease
  • CRISPR/Cas9 systems use different mechanisms to generate sequences.
  • DLBs double-strand breaks
  • These techniques are widely used in functional gene research [10], dynamic and real-time imaging of chromosomal loci [11,12], correction of disease mutations [13], gene therapy [14] and so on.
  • the CRISPR/Cas9 system has become particularly popular for its efficiency and ease of operation.
  • the CRISPR/Cas9 system was originally used by the bacterial immune system to protect against foreign viruses or plasmids.
  • Cas9 endonuclease cleaves double-stranded DNA under the guidance of sgRNA, resulting in genomic double-strand breaks, using cellular genome repair.
  • the instability causes a repair error (base deletion or insertion), which results in the effect of gene editing.
  • the CRISPR/Cas9 system has all of the previous advantages in design-based and sequence-specific genomic studies, the gene editing triggered by the CRISPR/Cas9 system is still a rare event in the cell population, and the real gene is edited.
  • the monoclonals require cumbersome labor, so the system is still technically challenging, even for simple tasks such as gene knockouts in mammalian cells [15].
  • gene knockout clones can be readily obtained, for example, Hela CSPG4 -/- cells confer resistance to Clostridium difficile toxin B [23] ].
  • Hela CSPG4 -/- cells confer resistance to Clostridium difficile toxin B [23] ].
  • this strategy cannot be generalized.
  • the traditional approach is to co-transfect plasmids expressing antibiotic resistance or fluorescent proteins [23, 24], but this method does not enrich a small number of cells containing targeted modifications.
  • exogenous dsDNA fragments may be integrated into chromosomal loci with DSBs by different repair mechanisms.
  • Reconstruction by homologous recombination (HR) requires the construction of long-flanking sequences with low integration efficiency, while the integration efficiency of non-homologous end joing (NHEJ) DNA repair is generally higher than homologous [27,28] Reorganization and repair [29].
  • NHEJ non-homologous end joing
  • the invention provides donor constructs, as well as gene knockout methods and systems and kits thereof.
  • the gene knockout method of the present invention enriches a gene-knock-out cell by using a marker gene contained in the donor construct, and improves the efficiency of gene knock-out by sequence-specific nuclease.
  • a donor construct which is linear donor DNA or which can be cleaved in a cell to produce linear donor DNA, the linear donor DNA comprising, in order from the middle to both ends An expression cassette; a short sequence extension consisting of a reverse stop codon at the 5' end of the expression cassette; and a short sequence extension consisting of a forward stop codon at the 3' end of the expression cassette; located at the 5' end and/or 3 a target sequence of the end, the target sequence comprising a target site cleavable by the sequence-specific nuclease; a protection sequence at both ends; wherein the expression cassette comprises a marker gene driven by a promoter.
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the donor construct is a linear donor DNA.
  • sequence specific nuclease is a zinc finger nuclease (ZFN).
  • sequence-specific nuclease is a transcriptional activator-like effector nuclease (TALEN).
  • TALEN transcriptional activator-like effector nuclease
  • sequence specific nuclease is a Cas9 nuclease.
  • sequence specific nuclease is an NgAgo nuclease.
  • the linear donor DNA has a target sequence only at the 5' or 3' end.
  • the linear donor DNA has a target sequence at each end.
  • the target sequences at both ends of the linear donor DNA are identical.
  • the target sequences at both ends of the linear donor DNA are different. In a further embodiment, the different target sequences at both ends of the linear donor DNA are derived from the same gene. In other further embodiments, the different target sequences at both ends of the linear donor DNA are derived from different genes.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the guard sequence is 5-30 bp in length, most preferably 20 bp.
  • a method of producing a gene knockout in a cell comprising the steps of:
  • the donor construct is a linear donor DNA or can be cleaved in a cell to produce a linear donor DNA comprising, in order from the middle to both ends: an expression cassette; the reverse of the 5' end of the expression cassette a short sequence extension consisting of a stop codon and a short sequence consisting of a forward stop codon at the 3' end of the expression cassette; a target sequence at the 5' end and/or the 3' end, the target sequence containing a sequence-specific nuclease-cleaved target site; a protection sequence at both ends; the expression cassette comprising a marker gene driven by a promoter;
  • linear donor DNA is inserted into a specific target site in the genome of the cell by non-homologous end joining
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the donor construct is a linear donor DNA.
  • the linear donor DNA has a target sequence only at the 5' or 3' end.
  • the linear donor DNA has a target sequence at each end.
  • the target sequences at both ends of the linear donor DNA are identical.
  • the target sequences at both ends of the linear donor DNA are different. In a further embodiment, the different target sequences at both ends of the linear donor DNA are derived from the same gene. In other further embodiments, the different target sequences at both ends of the linear donor DNA are derived from different genes.
  • sequence specific nuclease is a zinc finger nuclease (ZFN).
  • sequence-specific nuclease is a transcriptional activator-like effector nuclease (TALEN).
  • TALEN transcriptional activator-like effector nuclease
  • sequence specific nuclease is a Cas9 nuclease.
  • the method further comprises introducing into the cell a guide RNA (gRNA) that recognizes a particular target site in the genome of the cell, the target sequence in the linear donor DNA comprising a target site recognized by the gRNA.
  • gRNA guide RNA
  • the gRNA is an sgRNA.
  • the method further comprises introducing into the cell an sgRNA that recognizes a single specific target site in the genome of the cell, the target sequence comprising the target site recognized by the sgRNA being located 5' of the linear donor DNA End and / or 3' end.
  • the target sequence comprising the target site recognized by the sgRNA is derived from a single base in the genome of the cell because.
  • the target sequence comprising a target site recognized by the sgRNA is a consensus sequence of two or more genes in the genome of the cell, provided that the consensus sequence is associated with any of the two or more genes A sequence at a position corresponding to the consensus sequence has a difference of no more than one base.
  • the method further comprises introducing into the cell two sgRNAs that recognize two specific target sites on one gene in the genome of the cell, each comprising two recognized by the two sgRNAs
  • the two target sequences of the target site are located in two linear donor DNAs, respectively, or at the ends of the same linear donor DNA.
  • the method further comprises introducing into the cell two or more sgRNAs that recognize two or more specific target sites in the genome of the cell, respectively comprising the two or more Two or more target sequences of two or more target sites recognized by the sgRNA are located at both ends of the same linear donor DNA or in different linear donor DNAs, respectively. Wherein two or more specific target sites in the genome of the cell are located on different genes.
  • sequence specific nuclease is an NgAgo nuclease.
  • the method further comprises introducing into the cell a guide DNA (gDNA) introduced into a specific target site in the genome of the recognition cell, wherein the target sequence in the linear donor DNA comprises a target site recognized by the gDNA point.
  • gDNA guide DNA
  • the gene knockout may be a single gene knockout or a multiple gene knockout.
  • the polygenic knockout is a knockout of two or more genes, such as knockout of three, four, five or more genes.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the cells are screened by drug resistance.
  • the cells are screened by the FACS method.
  • the guard sequence is 5-30 bp in length, most preferably 20 bp.
  • a system or kit for gene knockout comprising: a sequence-specific nuclease and a donor construct capable of cleaving a specific target site in a genome of a cell;
  • the donor construct is a linear donor DNA or can be cleaved in a cell to produce a linear donor DNA comprising, in order from the middle to both ends: an expression cassette; the reverse of the 5' end of the expression cassette a short sequence extension consisting of a stop codon and a short sequence consisting of a forward stop codon at the 3' end of the expression cassette; a target sequence at the 5' end and/or the 3' end, the target sequence containing The sequence-specific nuclease cleavage target site; a protection sequence at both ends; the expression cassette comprising a marker gene driven by a promoter.
  • the linear donor DNA is a double-stranded linear donor DNA.
  • the donor construct is a linear donor DNA. In other embodiments, the donor construct is a circular donor construct and can be cleaved in a cell to produce a linear donor DNA.
  • sequence specific nuclease is a zinc finger nuclease (ZFN).
  • sequence-specific nuclease is a transcriptional activator-like effector nuclease (TALEN).
  • TALEN transcriptional activator-like effector nuclease
  • sequence specific nuclease is a Cas9 nuclease.
  • system or kit further comprises an sgRNA that recognizes a particular target site in the genome of the cell, wherein the target sequence in the linear donor DNA comprises a target site that is recognized by the sgRNA.
  • the gRNA is an sgRNA.
  • sequence specific nuclease is an NgAgo nuclease.
  • system or kit further comprises gDNA that recognizes a particular target site in the genome of the cell, wherein the target sequence in the linear donor DNA comprises a target site recognized by the gDNA.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the guard sequence is 5-30 bp in length, most preferably 20 bp.
  • the cleavage is the generation of double-strand breaks (DSBs).
  • a universal donor construct which is linear donor DNA or which can be cleaved in a cell to produce linear donor DNA from the middle to the two
  • the terminal comprises: an expression cassette; a short sequence extension consisting of a reverse stop codon at the 5' end of the expression cassette; and a short sequence extension consisting of a forward stop codon at the 3' end of the expression cassette; a universal target sequence at the 3' end, the universal target sequence comprising a target site cleavable by Cas9 nuclease; a protection sequence at both ends;
  • the expression cassette comprises a marker gene driven by a promoter
  • the universal target sequence is absent from the genome of the cell to be knocked out.
  • the universal donor construct is a linear donor DNA.
  • the linear donor DNA is a double stranded linear donor DNA.
  • the linear donor DNA has the universal target sequence only at the 5' or 3' end.
  • the linear donor DNA has the universal target sequence at both ends, respectively.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the guard sequence is 5-30 bp in length, most preferably 20 bp.
  • the universal target sequence in the universal donor construct contains 5'-GTACGGGGCGATCATCCACA-3' or 5'-AATCGACTCGAACTTCGTGT-3'.
  • a method of producing a gene knockout in a cell comprising the steps of:
  • a universal donor construct wherein the universal donor construct is linear donor DNA or can be cleaved in a cell to produce linear donor DNA comprising, in order from the middle to both ends: expression a short sequence extension consisting of a reverse stop codon at the 5' end of the expression cassette and a short sequence extension consisting of a forward stop codon at the 3' end of the expression cassette; at the 5' end and/or the 3' end a universal target sequence comprising a target site cleavable by Cas9 nuclease; a protection sequence at both ends;
  • the expression cassette comprises a marker gene driven by a promoter
  • the universal target sequence is absent from the genome of the cell to be knocked out
  • the donor construct is a linear donor DNA.
  • the linear donor DNA is a double stranded linear donor DNA.
  • the linear donor DNA has the universal target sequence only at the 5' or 3' end.
  • the linear donor DNA has the universal target sequence at both ends.
  • the gRNA recognizing a particular target sequence in the genome of the cell can be a gRNA, or a plurality of gRNAs that recognize different target sequences in the genome of the cell, eg, two, three or more recognition cell genomes gRNAs of different target sequences.
  • the different target sequences may be located in the same gene or may be located in different genes. Knockout of multiple genes can be achieved when the different target sequences are located in different genes, respectively.
  • the gene knockout may be a single gene knockout or a multi-gene knockout.
  • the polygenic knockout is a knockout of two or more genes, such as knockout of three, four, five or more genes.
  • the gRNA that recognizes a particular target sequence in the genome of a cell is an sgRNA.
  • the gRNA that recognizes a universal target sequence contained in a linear donor DNA is an sgRNA.
  • the sgRNA that recognizes a particular target sequence in the genome of the cell and the sgRNA that recognizes the universal target sequence contained in the linear donor DNA are located in the same vector.
  • the sgRNA that recognizes a particular target sequence in the genome of the cell and the sgRNA that recognizes the universal target sequence contained in the linear donor DNA are in separate vectors.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the cells are screened by drug resistance.
  • the cells are screened by the FACS method.
  • the guard sequence is 5-30 bp in length, most preferably 20 bp.
  • the universal target sequence in the universal donor construct contains 5'-GTACGGGGCGATCATCCACA-3' or 5'-AATCGACTCGAACTTCGTGT-3'.
  • a system or kit for gene knockout comprising:
  • a universal donor construct wherein the universal donor construct is linear donor DNA or can be cleaved in a cell to produce linear donor DNA, the linear donor DNA comprising: a short sequence extension consisting of a reverse stop codon at the 5' end of the expression cassette and a short sequence extension consisting of a forward stop codon at the 3' end of the expression cassette; at the 5' end and/or the 3' end a universal target sequence comprising a target site cleavable by Cas9 nuclease; a protection sequence at both ends;
  • the expression cassette comprises a marker gene driven by a promoter
  • the universal target sequence is absent from the genome of the cell to be knocked out
  • a gRNA that recognizes a universal target sequence contained in a linear donor DNA (4) A gRNA that recognizes a universal target sequence contained in a linear donor DNA.
  • the linear donor DNA is a double stranded linear donor DNA.
  • the donor construct is a linear donor DNA.
  • the donor construct is a circular donor construct and can be cleaved in a cell to produce a linear donor DNA.
  • the gRNA recognizing a particular target sequence in the genome of the cell can be a gRNA, or a plurality of gRNAs that recognize different target sequences in the genome of the cell, eg, two, three or more recognition cell genomes gRNAs of different target sequences.
  • the different target sequences may be located in the same gene or may be located in different genes. Knockout of multiple genes can be achieved when the different target sequences are located in different genes, respectively.
  • the gRNA that recognizes a particular target sequence in the genome of a cell is an sgRNA.
  • the gRNA that recognizes a universal target sequence contained in a linear donor DNA is an sgRNA.
  • the gRNA that recognizes a particular target sequence in the genome of the cell and the gRNA that recognizes the universal target sequence contained in the linear donor DNA are located in the same vector.
  • the gRNA identifying a particular target sequence in a cellular genome and the recognizing linear donor are located in different vectors.
  • the marker gene is an antibiotic resistance gene or a fluorescent protein gene.
  • the guard sequence is 5-30 bp in length, most preferably 20 bp.
  • the cleavage is the generation of double-strand breaks (DSBs).
  • the target sequence in the universal donor construct contains 5'-GTACGGGGCGATCATCCACA-3' or 5'-AATCGACTCGAACTTCGTGT-3'.
  • the present invention can efficiently enrich a rare clone having a gene knockout by inserting a marker gene into a gene-cleaved cleavage target site.
  • the present invention is particularly useful for the targeting of genes that are difficult to design sgRNAs, as well as where it is desirable to simultaneously target several gene knockouts. This method is useful for a variety of gene editing systems that generate DNA double-strand breaks, especially the wider application of the CRISPR system in the biomedical field of genes and their functions.
  • Figure 1 is a donor design and experimental validation of cells containing Cas9/gRNA targeting mutations in the ANTXR1 gene enriched in HeLa cells by puromycin selection.
  • Figure 2 is an experimental validation of enrichment of ANTXR1 knockout cells in mixed colonies and monoclonals by donor-mediated puromycin resistance selection.
  • (a) Images of different HeLa cell groups treated with or without PA/LFnDTA. Mixed cells were obtained with sgRNA or pgRNA with or without their corresponding linear donors, with a scale of 200 ⁇ m.
  • Figure 3 is a donor design and experimental validation of HEBGF destruction events in HeLa cells enriched by puromycin.
  • Donor design targeting the HBEGF gene (a) Donor design targeting the HBEGF gene.
  • sgRNA1 HBEGF sgRNA1 HBEGF
  • Donor HBEGF-sg1 with or without the corresponding linear donor
  • sgRNA1 HBEGF sgRNA1 HBEGF
  • a plasmid expressing Cas9 a reporter plasmid containing a puromycin resistance gene (light bar)
  • a sgRNA, a plasmid expressing Cas9 and a linear donor (Donor HBEGF-sg1 )
  • Figure 4 is a donor design and experimental validation of HBEGF disruption events in HEC293T cells enriched with EGFP.
  • sgRNA2 HBEGF sgRNA2 HBEGF
  • Donor HBEGF-sg2 linear donor
  • sgRNA2 HBEGF sgRNA2 HBEGF plasmid expressing mCherry and Cas9-expressing plasmid (light bar), or sgRNA, Cas9-expressing plasmid and linear donor (Donor HBEGF-sg2 , EGFP) (dark bars)
  • Figure 5 is a donor design and experimental validation of the ANTXR1 disruption event in HeLa OC cells enriched by puromycin.
  • Figure 6 is a off-target assessment of donor insertion by splinkerette PCR (spPCR) analysis in HeLa OC cells.
  • spPCR splinkerette PCR
  • Figure 7 is a donor design and experimental validation of HBEGF disruption events in HeLa OC cells enriched by puromycin.
  • Donor design targeting HBEGF The donor contains an sgRNA cleavage site (Donor HBEGF-sg1 or Donor HBEGF-sg2 ) at the 5' end or two gRNAs (Donor HBEGF-pg ) at both ends.
  • Donor HBEGF-sg1 or Donor HBEGF-sg2 MTT staining of puromycin resistant clones in cells transfected with donor (with or without sgRNA/pgRNA).
  • Figure 8 is a donor design and experimental validation of one or more gene knockouts in one step in HeLa OC cells.
  • Clone 1 (b) was derived from HeLa OC cells transfected with pgRNA PSEN1 + PSEN2 / Donor PSEN1 + PSEN2 .
  • Clone 2 (c) was derived from HeLa OC cells transfected with pgRNA PSEN1 + PSEN2 / Donor PSEN1 + Donor PSEN2 .
  • the nucleotides in the shaded region represent PAM sequences that direct Cas9 for DNA recognition and cleavage.
  • the dotted line indicates the deletion, the high letter indicates the nucleotide insertion, and the light gray arrow in the background indicates the direction of the CMV promoter in the donor.
  • Black shaded nucleotides represent the consensus sequence of all five HSPA genes. Dark gray shaded nucleotides represent the consensus sequence of three or four HSPA genes, and light gray shaded nucleotides represent non-consensus nucleotides.
  • Indels primed by sgRNA HSPA on five target genes following puromycin selection in the absence and presence of Donor HSPA . Error bars indicate sd(n 3), t-test, **P ⁇ 0.01, ***P ⁇ 0.001.
  • Clone 3 was derived from HeLa OC cells transfected with sgRNA HSPA /Donor HSPA .
  • the nucleotides in the shaded portion represent the PAM sequence and the dashed lines represent the deletion.
  • the light gray arrows in the background indicate the direction of the CMV promoter in the donor.
  • Figure 9 is a PSEN1 and PSEN2sgRNA efficiency evaluation and single clone recognition in HeLa OC cells.
  • Figure 10 is a sequencing view of the target region of the HSPA family gene in mixed cells transfected with or without donor.
  • the sgRNA target sites are shaded and do not include target regions containing donor insertions in these sequencing analyses.
  • Figure 11 is a monoclonal identification of donor insertions at the target sites of five HSPA family genes, HSPA1A, HSPA1B, HSPA1L, HSPA6 and HSPA2.
  • HSPA1A, HSPA1B, HSPA1L, HSPA6 and HSPA2. (a) A linear donor-integration of puromycin-resistant monoclonal at all five loci PCR verification results.
  • Figure 12 is an experimental flow diagram of gene knockout using a donor comprising a universal sgRNA.
  • Figure 13 is a gene knockout validation of gene knockout using a donor comprising a universal sgRNA.
  • the present invention provides novel donor constructs and gene knockout methods that utilize linear donor DNA to increase the efficiency of gene knockout by sequence-specific nucleases.
  • the linear donor DNA of the present invention comprises at least one target site that can be cleaved by a sequence-specific nuclease.
  • the target site contained in the linear donor DNA is designed based on the target site in the genome of the cell, such that a sequence-specific nuclease capable of cleaving a target site in the genome of the cell can also cleave a target site contained in the linear donor DNA.
  • sequence-specific nuclease and a donor construct When a sequence-specific nuclease and a donor construct are introduced into a cell, the sequence-specific nuclease simultaneously cleaves at least one target contained in the linear donor DNA when a double-strand break (DSBs) is generated at a specific target site in the cell. Point, thereby allowing linear donor DNA to be inserted into the cleavage target site of the cell genome by a non-homologous end joining (NHEJ) pathway with higher efficiency, followed by the marker Selection of cells can efficiently enrich cells that are knocked out by cleavage at specific target sites in the genome, greatly increasing the efficiency of gene knockout by sequence-specific nucleases.
  • NHEJ non-homologous end joining
  • the target site contained in the linear donor DNA is designed based on the target site in the genome of the cell, and the linear donor DNA obtained is a specific linear donor when gene knockout is required at different target sites in the genome of the cell. It is necessary to construct a linear donor DNA for use according to the sequence of the target site.
  • the inventors further provide in the present invention a universal linear donor DNA comprising a universal target sequence cleavable by a sequence-specific nuclease, the universal target sequence being The genome of the knock-out cell is absent, i.e., the same sequence as the universal target sequence cleavable by the sequence-specific nuclease is absent from the genome of the cell to be knocked out.
  • the sequence-specific nuclease when a sequence-specific nuclease and a universal linear donor DNA are introduced into a cell, the sequence-specific nuclease generates a double-strand break (DSBs) at a specific target site in the cell, and is contained in the universal linear donor DNA.
  • the universal target sequence is also cleaved by the sequence-specific nuclease by a universal gRNA that recognizes the target sequence, at which time the linear donor DNA can still pass the Non-Homologous end joining (NHEJ) pathway with higher efficiency.
  • NHEJ Non-Homologous end joining
  • the earth increases the efficiency of gene knockout by sequence-specific nucleases.
  • the target sequence in the universal linear donor DNA is independent of the gene to be knocked out, and it can be used as a universal The use of donors for knockout of different target genes in different cells can increase the efficiency of gene knockout by sequence-specific nucleases.
  • Universal linear donor DNA is particularly useful for gene knockouts using the Cas9/CRISPR system, which uses gRNA (preferably sgRNA) to target a target sequence, and when gene knockout, only needs to be mapped to a specific target in the genome of the cell.
  • gRNA preferably sgRNA
  • the point gRNA eliminates the need to specifically construct a linear donor DNA for use, and directly uses the universal linear donor DNA and the gRNA targeting the universal linear donor DNA, thereby reducing the operational complexity and improving the efficiency.
  • the mutation frequency of the target allele is usually higher [25, 26]. Therefore, although not wishing to be bound by theory, the inventors speculate that if a donor can be inserted at a specific site on one of the target alleles and a clone expressing the marker gene contained in the donor is selected, it may be possible to enrich. Rare events where all alleles are modified.
  • knockout is the loss of gene function by gene editing.
  • the gene knockout effect usually pursued is that both alleles are knocked out at the same time, and at this time, the corresponding protein loses its function, and a knockout cell line is obtained. If only one allele is knocked out, the protein can also play a part, only the downregulation of the protein. With the linear donor DNA of the present invention and the method of the present invention, cells in which both alleles are knocked out can be efficiently enriched.
  • the donor construct of the invention is a double stranded DNA.
  • the donor construct of the invention may itself be a linear donor DNA.
  • the donor construct of the invention may be a circular DNA molecule comprising linear donor DNA that, when introduced into a cell, is cleaved in the cell to produce a linear donor DNA.
  • Methods of cleavage of circular donor constructs in cells to produce linear donor DNA are well known in the art.
  • a cleavage site for another sequence-specific nuclease can be further included in the circular construct upstream of the 5' end and downstream of the 3' end of the linear donor DNA.
  • it may further comprise introducing into the cell another sequence-specific nuclease which cleaves the 5' upstream and the 3' downstream of the linear donor DNA in the circular construct in the cell.
  • the sequence, resulting in linear donor DNA may further comprise introducing into the cell another sequence-specific nuclease which cleaves the 5' upstream and the 3' downstream of the linear donor DNA in the circular construct in the cell. The sequence, resulting in linear donor DNA.
  • reverse stop codon means that the direction of the codon is opposite to the orientation of the reading frame of the expression cassette.
  • Positive termination codon means that the direction of the codon is the same as the orientation of the reading frame of the expression cassette. The role of the stop codon is that both triple stop codons can stop endogenous and exogenous gene expression regardless of whether the linear donor is inserted into the genome in either the forward or reverse direction.
  • the "protection sequence" in the linear donor DNA of the present invention may be any sequence, and preferably the protection sequence is different from the target sequence in the same linear donor DNA.
  • the length of the protection sequence may be 5-30 bp, preferably 20 bp.
  • the role of the protection sequence is to protect the target sequence in the linear donor DNA from being cleaved by an enzyme (eg, an exonuclease) in the cell.
  • a "marker gene” refers to any marker gene whose expression can be selected or enriched, ie when When the marker gene is expressed in a cell, the cell expressing the marker gene can be selected and enriched in a certain manner.
  • Marker genes useful in the present invention include, but are not limited to, fluorescent protein genes which can be sorted by FACS after expression, or resistance genes which can be screened by antibiotics, or which can be recognized by corresponding antibodies and expressed by immunostaining or magnetic The beads adsorbed the protein gene for screening.
  • Resistant genes useful in the present invention include, but are not limited to, against blasticidin (Blasticidin), geneticin (G-418), hygromycin B, Mycophenolic Acid, guanidine.
  • Fluorescent protein genes useful in the present invention include, but are not limited to, Cyan Fluorescent Protein, Green Fluorescent Protein, Yellow Fluorescent Protein, Orange Fluorescent Protein, Red Genes for Red Fluorescent Protein, Far-Red Fluorescent Protein, or Switchable Fluorescent Proteins.
  • sequence-specific nucleases include zinc finger nucleases (ZFNs).
  • Zinc finger nucleases are non-naturally occurring, artificially engineered endonucleases consisting of a zinc finger protein domain and a non-specific endonuclease domain.
  • the zinc finger protein domain consists of a series of Cys2-His2 zinc finger proteins in tandem, each zinc finger protein recognizing and binding to a specific triplet base on the 3' to 5' direction DNA strand and one in the 5' to 3' direction Base. Multiple zinc finger proteins can be cascaded to form a zinc finger protein group that recognizes a specific base sequence with strong specificity.
  • the non-specific endonuclease linked to the zinc finger protein group is derived from the DNA cleavage domain consisting of 96 amino acid residues at the carboxy terminus of FokI.
  • Each FokI monomer is linked to a zinc finger protein group to form a ZFN, which recognizes a specific site.
  • the two recognition sites are at an appropriate distance (6-8 bp)
  • the two monomeric ZFN interactions produce a digestive function.
  • Designing 8 to 10 zinc finger domains for the target sequence, and linking these zinc domains to DNA nucleases can achieve double strand breaks (DSBs) of the target sequence, and then induce DSBs repair mechanism to the genome. Orientation modification at a specific site in the process.
  • sequence specific nuclease includes a transcriptional activator-like effector nuclease (TALEN).
  • the transcriptional activator-like effector nuclease is mainly composed of a Fok I endonuclease domain and a DNA binding domain of a TALE protein.
  • the TALE protein contains multiple repeating peptides of 33-35 amino acids, and each peptide recognizes one base.
  • TALEN can also cleave DNA target sequences to form DSBs, thereby activating DNA damage repair mechanisms and performing site-specific transformation of the genome.
  • Cas9/CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the Cas9/CRISPR system utilizes RNA-directed DNA binding for sequence-specific cleavage of target DNA, and by crRNA (CRISPR-derived RNA) base pairing with tracrRNA (trans-activating RNA)
  • crRNA CRISPR-derived RNA
  • tracrRNA trans-activating RNA
  • a tracrRNA/crRNA complex that directs the nuclease Cas9 protein to cleave double-stranded DNA at a specific position on the target sequence that is paired with the crRNA.
  • the target sequence paired with the crRNA is typically a sequence of about 20 nucleotides upstream of the genomic PAM (proximal spacer adjacent motif) site (NNG).
  • NNG genomic PAM
  • the Cas9 protein cleaves the target site by means of a guide RNA.
  • guide RNA is also known as gRNA (guide RNA).
  • GRNA usually includes a nucleotide complementary to a target sequence on a crRNA and an RNA scaffold formed by base pairing of a crRNA and a tracrRNA, and is capable of recognizing a target sequence paired with a crRNA. .
  • the gRNA can form a complex with the Cas9 protein and bring the Cas9 protein to the target sequence and cleave the target site therein.
  • gRNA is usually used in the form of sgRNA (single guide RNA).
  • sgRNA also known as “single-stranded guide RNA”
  • crRNA RNA strand fused with crRNA and trancrRNA.
  • NgAgo nuclease Another example of a sequence-specific nuclease system useful in the present invention includes NgAgo nuclease and its gDNA.
  • the NgAgo nuclease can bind to the 5'-terminally phosphorylated single-stranded guide DNA (gDNA) to cleave the target sequence complementary to the gDNA, resulting in DNA double-strand breaks.
  • gDNA 5'-terminally phosphorylated single-stranded guide DNA
  • the linear donor DNA of the present invention may have a target sequence only at one end, or may have a target sequence at both ends, respectively.
  • the target sequences at both ends of the linear donor DNA can be different.
  • two linear donor DNAs can be provided, each containing a corresponding target sequence, or a linear Donor DNA, each end of the linear donor DNA comprising a corresponding target sequence.
  • an appropriate amount of linear donor DNA can be provided, and each linear donor DNA contains a plurality of different corresponding targets at one or both ends One of the sequences.
  • linear donor DNA of the same number as the target site can be provided, each linear donor DNA comprising a respective target sequence.
  • linear donor DNA may be provided in an amount less than the number of target sites, wherein all or part of the linear donor DNA contains one of a plurality of different respective target sequences, and the other linear donor DNAs each contain other corresponding targets.
  • the universal target sequence can be contained at either or both ends thereof, and the target sequence of the universal linear donor DNA is independent of the target site to be excised in the cell genome.
  • the point thus, can be universally applied to the case where a gene knockout is produced by cleavage of any one of the target sites, any two target sites, or any more of the target sites in the genome of the cell.
  • the "universal target sequence” as used in the present invention refers to a sequence which can be cleaved by a sequence-specific nuclease, but the universal target sequence does not exist in the genome of the cell to be subjected to gene knockout, that is, the gene to be subjected to
  • the sequence of the knockout cell does not have the same sequence as the universal target sequence cleavable by the sequence-specific nuclease, and the universal target sequence and the target present on the genome of the cell that can be cleaved by the same sequence-specific nuclease
  • the sequences are all different.
  • Line containing the universal target site Sex donor DNA is not specific for the target site on the genome of the cell, so it can be universally applied to gene knockout of any gene in the cell without constructing a specific target for the gene to be knocked out Linear donor DNA.
  • sequence specific nuclease can be introduced into the cell in the form of a protein or in the form of its encoding nucleic acid sequence (eg, mRNA or cDNA).
  • a nucleic acid encoding a sequence-specific nuclease can be introduced into a cell, or introduced into a cell, for example by transfection, in a plasmid or viral vector.
  • Nucleic acids encoding sequence-specific nucleases can also be delivered directly to cells by electroporation, liposome, microinjection, and the like.
  • the donor construct can be delivered by any method suitable for introducing the nucleic acid into the cell, such as by transfection into a cell.
  • sgRNA or gDNA is also introduced into the cells.
  • the sgRNA or gDNA can be delivered by any method suitable for introducing RNA or DNA into the cell.
  • the sgRNA can be introduced into the cell in the form of isolated RNA. Isolated sgRNA can be prepared by in vitro transcription using any in vitro transcription system known in the art.
  • the sgRNA can also be introduced into the cell by a vector comprising a sequence encoding the sgRNA and a promoter.
  • the vector may be a viral vector or a plasmid.
  • the means of introducing the cells can be transfection.
  • Two or more sgRNAs directed to different target sites, respectively, can be introduced into the cell to direct cleavage at two or more different target sites in the Cas9 cleavage cell genome to generate a gene knockout.
  • the two or more sgRNAs may be contained in different vectors, or may be contained in the same vector, such as a vector comprising a pair of gRNAs (paired gRNA), or a vector containing more sgRNA.
  • linear donor DNA comprising a target sequence recognized by these sgRNAs is simultaneously introduced. Since the linear donor DNA may contain the target sequence only at the 5' or 3' end, it may also contain the target sequence at both ends, and the number of sgRNAs and the number of linear donor DNAs may be different, and one sgRNA may be a linear one.
  • the bulk DNA may also be two sgRNAs corresponding to two linear donor DNAs.
  • sgRNA an sgRNA directed against a universal target sequence on universal linear donor DNA, to direct Cas9 to cleave specific target sequences in the genome of the cell and universal target sequences on universal linear donor DNA.
  • the sgRNA directed against a specific target sequence in the genome of the cell and the sgRNA directed against a universal target sequence on a universal linear donor DNA may be contained in different vectors or may be contained in the same vector.
  • the sgRNA directed against a specific target sequence in the genome of the cell may be an sgRNA or more sgRNAs, for example, two species, 3 or more gRNAs. These more than one sgRNA can be directed to different specific target sequences in the genome of the cell, respectively, to effect simultaneous cleavage of different target sites on the genome of the cell. Knockout of multiple genes, such as knockout of two, three or more genes, can be achieved when these different target sites are located on different genes, respectively.
  • a plurality of sgRNAs directed against a plurality of specific target sequences in a cell genome and sgRNAs directed against a universal target sequence on a universal linear donor DNA can be introduced into the cells to guide Cas9 Multiple specific target sequences in the genome of the cell and universal target sequences on the universal linear donor DNA are cleaved.
  • the plurality of specific target sequences are respectively located on different genes, thereby achieving multi-gene knockout.
  • the plurality of sgRNAs directed to a plurality of specific target sequences in the genome of the cell, respectively, may be contained in different vectors or may be contained in the same vector.
  • the sgRNA of any one or more sgRNAs directed to a plurality of specific target sequences in the genome of the cell and the universal target sequence on the universal linear donor DNA may be contained in different vectors, Can be included in the same carrier.
  • the universal target sequence on the universal linear donor DNA is preferably 5'-GTACGGGGCGATCATCCACA-3' or 5'-AATCGACTCGAACTTCGTGT-3'.
  • Cas9, sgRNA and linear donor DNA can be simultaneously introduced into the cell, or, for example, Cas9 can be introduced into the cell first, followed by sgRNA and Linear donor DNA is introduced into the cell.
  • the cells are co-transfected with a vector comprising Cas9, a vector comprising sgRNA, and linear donor DNA.
  • Cas9 and sgRNA are assembled in vitro into protein and RNA complexes and co-transfected with linear donor DNA.
  • Cas9 and sgRNA are stably expressed into the cell by lentivirus and the cells are transfected with linear donor DNA.
  • Cas9 is first stably expressed in cells, and the cells are co-transfected with a vector comprising sgRNA and linear donor DNA.
  • the sequence-specific nuclease may be in the form of a protein or a nucleic acid sequence (eg, mRNA or cDNA), for example, comprising a coding sequence-specific nuclease.
  • the nucleic acid is in the form of a plasmid or viral vector.
  • the sgRNA may be in the form of an isolated RNA or a vector comprising a sequence encoding the sgRNA and a promoter, such as a viral vector or a plasmid vector.
  • the cells described herein can be any eukaryotic cell, such as an isolated animal cell, such as a totipotent cell, a pluripotent cell, an adult stem cell, a fertilized egg or a somatic cell, and the like.
  • the cell is a vertebrate cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a cell of a cow, goat, sheep, cat, dog, horse, rodent, fish, primate.
  • the rodent comprises mouse, rat, rabbit.
  • the methods of the invention can be used to target gene knockouts on a single gene or genes in a cell, for example targeting two, three, four, five or more gene knockouts.
  • Targeted gene knockouts for multiple genes can be performed simultaneously or sequentially.
  • a sequence-specific nuclease or sequence-specific nuclease system for two or more target genes can be introduced into a cell and then subjected to enrichment screening.
  • a sequence-specific nuclease or sequence-specific nuclease system directed against one or more target genes can be introduced into a cell and subjected to enrichment screening, followed by sequence-specific nucleases or sequences specific for other target genes.
  • the nuclease system is introduced into the cells and subjected to enrichment screening.
  • Different marker/marker genes can be used for different target genes.
  • two or more sgRNAs directed to different target sites can be introduced into the cell, and a linear supply containing the target sequence recognized by these sgRNAs is simultaneously introduced. Body DNA, as described above. When these different target sites are located on different genes, gene knockout of multiple genes can be achieved.
  • a target sequence in sgRNA and linear donor DNA by using a consensus sequence of two or more genes in the genome of the cell, in which case sgRNA recognizing a single specific target site in the genome of the cell can be introduced into the cell, and the inclusion is included a linear donor DNA of a target sequence recognized by the sgRNA, wherein the target sequence recognized by the sgRNA is a consensus sequence of two or more genes in a genome of the cell, provided that the consensus sequence is identical to the two or more
  • the sequence at any position corresponding to the consensus sequence on any one of the genes has a difference of no more than one base. A difference in two bases may disrupt the recognition of sgRNA as demonstrated in Example 7.
  • the target gene to which the gene editing by the linear donor DNA of the present invention is directed is not particularly limited as long as it can generate a double-strand break by the Cas9/CRISPR system.
  • the target gene can be an exon, an intron or a regulatory sequence, or any combination thereof.
  • sgRNAs targeting the first exon of the ANTXR1 gene in HeLa cells were designed and verified for their efficiency of deletion or insertion mutation (Indels) at the target site by T7E1 assay.
  • the results are shown in Table 1.
  • SgRNA1 ANTXR1 wherein the target sequence in the present embodiment for example is referred sg1, sgRNA2 ANTXR1 target sequence in the present embodiment for example is referred sg2.
  • Donor ANTXR1-sg2 includes a 20 bp protection sequence, a sg2, a reverse stop codon, a CMV promoter-driven puromycin resistance gene, a forward stop codon, and a 20 bp protection sequence from the 5' to the 3' end, respectively.
  • Donor ANTXR1-pg includes: 20bp protection sequence, sg1, reverse stop codon, CMV promoter-driven puromycin resistance gene, positive stop codon, sg2, 20bp protection from 5' to 3' sequence.
  • the linear donor DNA (Donor no cut ) as a control includes a 20 bp protection sequence, a 20 bp random sequence, a reverse stop codon, and a CMV promoter-driven puromycin resistance gene from the 5' to the 3' end, respectively. Positive stop codon, 20 bp protection sequence.
  • the random sequence is different from sg1 or sg2.
  • HeLa cells were co-transfected with a plasmid expressing Cas9, sgRNA2 ANTXR1 or pgRNA ANTXR1 , and their corresponding donors, and transfected with linear donor DNA (Donor ANTXR1-sg2 , Donor ANTXR1-pg and Donor no cut ) alone as a control HeLa cells were added with puromycin for resistance screening to obtain pooled populations and single clones using MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-Diphenyl bromide tetrazolium) staining, the results are shown in Figure 1b.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2, 5-Diphenyl bromide tetrazolium
  • a number of cell clones with puromycin resistance were obtained from sgRNA2 ANTXR1 and its corresponding donor Donor ANTXR1-sg2 , as well as from samples receiving pgRNA ANTXR1 and its corresponding donor Donor ANTXR1-pg . Only donor-transfected clones produced only a few puromycin-resistant clones, probably because the integration of linear donors onto the chromosome was sparse and random.
  • the Cas9-expressing plasmid and sgRNA2 ANTXR1 or pgRNA ANTXR1 were used , and the corresponding donors were co-transfected into HeLa cells.
  • the mixed community and monoclonal were screened by puromycin (1 ⁇ g/ml), without corresponding The donor was co-transfected with a plasmid expressing a puromycin resistance gene.
  • the ANTRX1 site of the puro+ monoclonal linear donor integration was subjected to PCR verification, and the L1/R1 primer sequence used in the PCR amplification is shown in Table 2.
  • the results are shown in Figure 2b, and it can be seen that most of the clones isolated from the puro+ cell mixture contain a donor insert at the sgRNA targeting site (see also Figure Id). It can also be seen from Figure 1d that nearly 90% of the cells carrying the donor fragment are true knockout clones.
  • a donor having a single- or double-cleavage site is capable of greatly increasing the selection of cells having modifications at the target site, for convenience, in the present embodiment, only a single-cut donor is used.
  • SgRNA1 HBEGF wherein the target sequence in the present embodiment for example is referred sg1, sgRNA2 HBEGF target sequence in the present embodiment for example is referred sg2.
  • a linear donor DNA (Donor HBEGF-sg1 ) was constructed, the structure of which is shown in Figure 3a.
  • Donor HBEGF-sg1 includes a 20 bp protection sequence, sg1, a reverse stop codon, a CMV promoter-driven puromycin resistance gene, a forward stop codon, and a 20 bp protection sequence from the 5' to the 3' end, respectively.
  • HeLa cells were co-transfected with Cas9-expressing plasmid, sgRNA1 HBEGF , and its corresponding donor Donor HBEGF-sg1 .
  • sgRNA1 HBEGF Cas9-expressing plasmid
  • donor Donor HBEGF-sg1 alone, and puromycin was added for resistance screening.
  • the pooled population and single clones were obtained and stained with MTT. The results are shown in Figure 3b.
  • the Cas9-expressing plasmid and sgRNA1 HBEGF were used , and the corresponding donor Donor HBEGF-sg1 was added to co-transfect HeLa cells, and the mixed community and monoclonal were screened by puromycin (1 ⁇ g/ml).
  • the corresponding donor was co-transfected with a plasmid expressing a puromycin resistance gene. Since the HBEGF gene encodes a diphtheria toxin (DT) receptor, knocking it out in HeLa cells results in cell resistance to DT [17], and mixed communities and singles obtained by treatment with DT (40 ng/ml) for puromycin. Cloning to compare the effect of linear donor DNA on HBEGF knockout efficiency.
  • DT diphtheria toxin
  • HBEGF knockout efficiency was determined by calculating the percentage of cells with DT resistance in the puro+ mixed community, as shown in Figure 3d.
  • Figures 3c and 3d the use of linear donor DNA greatly improved the HBEGF gene knockout efficiency compared to sgRNA1 HBEGF alone.
  • the sgRNA2 HBEGF targeting HBEGF gene in HEK293T cells was designed and the linear donor DNA (Donor HBEGF-sg2 ) was constructed.
  • the donors from the 5' to the 3' end include: 20 bp protection sequence, sg2, reverse stop codon, The CMV promoter-driven EGFP gene, a positive stop codon, a 20 bp protection sequence, see Figure 4a.
  • HEK293T cells were co-transfected with Cas9-expressing plasmid and sgRNA2 HBEGF with or without the corresponding donor Donor HBEGF-sg2 , and cells were screened by FACS.
  • the donor-derived group was screened for EGFP-positive cells by FACS without donor.
  • FACS-selected cells were treated with DT (40 ng/ml) to compare the effect of linear donor DNA on HBEGF knockout efficiency.
  • the images of different cells treated with DT are shown in Figure 4b.
  • HBEGF knockout efficiency was determined by calculating the percentage of cells with DT resistance in EGFP positive cells, as shown in Figure 4d.
  • the use of linear donor DNA greatly increased the efficiency of HBEGF gene knockout compared to sgRNA2 HBEGF alone.
  • the HeLa OC cell line [17] was established according to the existing method, and the cell line stably expressed Cas9.
  • SgRNA1 ANTXR1 wherein the target sequence in the present embodiment for example is referred sg1, sgRNA2 ANTXR1 target sequence in the present embodiment for example is referred sg2.
  • Donor ANTXR1-sg1 includes a 20 bp protection sequence, sg1, a reverse stop codon, a CMV promoter-driven puromycin resistance gene, a forward stop codon, and a 20 bp protection sequence from the 5' to the 3' end, respectively.
  • Donor ANTXR1-sg2 includes a 20 bp protection sequence, a sg2, a reverse stop codon, a CMV promoter-driven puromycin resistance gene, a forward stop codon, and a 20 bp protection sequence from the 5' to the 3' end, respectively.
  • Donor ANTXR1-pg includes: 20bp protection sequence, sg1, reverse stop codon, CMV promoter-driven puromycin resistance gene, positive stop codon, sg2, 20bp protection from 5' to 3' sequence.
  • HeLa OC cells were co-transfected with Cas9-expressing plasmid, sgRNA1 ANTXR1 or sgRNA2 ANTXR1 or pgRNA ANTXR1 , and their corresponding donors, as controls, using linear donor DNA alone (Donor ANTXR1-sg1 , Donor ANTXR1-sg2, and Donor ANTXR1) -pg ) HeLa OC cells were transfected, and puromycin was added for resistance screening and stained with MTT. The results are shown in Figure 5b.
  • HeLa OC cells were co-transfected with a plasmid expressing Cas9 and sgRNA1 ANTXR1 or sgRNA2 ANTXR1 or pgRNA ANTXR1 with or without their corresponding donors, and screened with puromycin (1 ⁇ g/ml).
  • the cells obtained by screening were treated with PA/LFnDTA, and the images of different cells treated with PA/LFnDTA are shown in Fig. 5c.
  • the ANTXR1 knockout efficiency was determined by calculating the percentage of cells with this toxin resistance in the puro+ mixed population, as shown in Figure 5d, and the use of linear donor DNA greatly improved the ANTXRl gene knockout efficiency compared to sgRNA alone.
  • PCR analysis of the integration site of the donor Donor ANTXR1-sg1 on the ANTXR1 gene in a single clone of puro+ revealed that most of the puro+ clones contained a donor insert at the sgRNA targeting site (Fig. 5e and Fig. 5f). Most of the cells carrying the donor fragment are true knockout clones (Fig. 5f).
  • a PCR fragment of ⁇ 500 bp (length corresponding to the wild type ANTXR1 gene) and a PCR fragment of ⁇ 1.8 kb (length corresponding to the wild type ANTXR1 gene plus donor insert) were subjected to genome sequencing, and the results are shown in Table 4.
  • Target site ANTXR1 (Chr2, HeLa oc )
  • the off-target insertion in single clones and mixed cell clones was verified by splinkerette PCR analysis after puromycin selection. If the correct donor insertion is made on the ANTXR1 gene, amplification with primers Splink2/R1 and Splink2/R2 will result in 711- and 927-bp products, respectively (see Figure 6a).
  • sgRNA1 HBEGF and sgRNA2 HBEGF targeting the HBEGF gene in HeLa OC cells were designed and three linear donor DNAs (Donor HBEGF-sg1 , Donor HBEGF-sg2 and Donor HBEGF-pg ) were constructed, see Figure 7a.
  • SgRNA1 HBEGF wherein the target sequence in the present embodiment for example is referred sg1, sgRNA2 HBEGF target sequence in the present embodiment for example is referred sg2.
  • Donor HBEGF-sg1 includes a 20 bp protection sequence, sg1, a reverse stop codon, a CMV promoter-driven puromycin resistance gene, a forward stop codon, and a 20 bp protection sequence from the 5' to the 3' end, respectively.
  • Donor HBEGF-sg2 includes a 20 bp protection sequence, sg2, a reverse stop codon, a CMV promoter-driven puromycin resistance gene, a forward stop codon, and a 20 bp protection sequence from the 5' to the 3' end, respectively.
  • Donor HBEGF-pg includes: 20 bp protection sequence, sg1, reverse stop codon, CMV promoter-driven puromycin resistance gene, positive stop codon, sg2, 20 bp protection from 5' to 3' end sequence.
  • HeLa OC cells were co-transfected with Cas9-expressing plasmid, sgRNA1 HBEGF or sgRNA2 HBEGF or pgRNA HBEGF , and their corresponding donors as controls, using linear donor DNA alone (Donor HBEGF-sg1 , Donor HBEGF-sg2, and Donor HBEGF) -pg ) HeLa OC cells were transfected, and puromycin was added for resistance screening. The results are shown in Figure 7b.
  • the integration site of donor Donor HBEGF-sg1 on the HBEGF gene was verified by PCR, and the L2/R2 primer sequence used in PCR amplification is shown in Table 5.
  • Most of the clones from puro+ were found to contain a donor insert at the sgRNA targeting site (Fig. 7c and Fig. 7d), and most of the cells carrying the donor fragment were true knockout clones (Fig. 7d).
  • Two target genes in HeLa OC cells PSEN1 and PSEN2 were selected, and two sgRNAs targeting the two target genes were designed to verify their efficiency in producing Indels at the target site by T7E1 assay. The results are shown in Table 6. Show.
  • the target sequence to which sgRNA PSEN1 is directed is referred to as sg PSEN1 in this example, and the target sequence to which sgRNA PSEN2 is directed is referred to as sg PSEN2 in this embodiment.
  • Donor PSEN1 + Donor PSEN2 Two types of donors were constructed, one with two separate donors (Donor PSEN1 + Donor PSEN2 ), each donor having a target sequence for the corresponding sgRNA, and the other donor (Donor PSEN ) having Two sgRNA target sequences are shown in Figure 8a.
  • Donor PSEN1 or Donor PSEN2 has a sgRNA PSEN1 or sgRNA PSEN2 cleavage site at its 5' end.
  • Donor PSEN1 + PSEN2 'having sgRNA PSEN1 terminal cleavage site at the 3' 5 having sgRNA PSEN2 cleavage site ends. among them:
  • Donor PSEN1 includes: 20 bp protection sequence, sg PSEN1 , reverse stop codon, CMV promoter driven puromycin resistance gene, positive stop codon, 20 bp protection sequence from 5' to 3' end.
  • Donor PSEN2 includes from the 5' to the 3' end: 20 bp protection sequence, sg PSEN2 , reverse stop codon, CMV promoter driven puromycin resistance gene, positive stop codon, 20 bp protection sequence.
  • Donor PSEN includes: 20bp protection sequence, sg PSEN1 , reverse stop codon, CMV promoter-driven puromycin resistance gene, forward stop codon, sg PSEN2 , 20bp protection from 5' to 3' sequence.
  • the HSPA gene family in HeLa OC cells was selected, which has five genes, HSAPA1A, HSPA1B, HSBA1L, HSPA6 and HSPA2, which share homology.
  • the sgRNA HSPA targeting both HSAPA1A, HSPA1B and HSBA1L was designed.
  • the target sequence of the sgRNA has a mismatch with the corresponding sequence on HSPA6 and two mismatches with HSPA2. As shown in Figure 8d.
  • HeLa OC cells were co-transfected with Cas9-expressing plasmid and sgRNA HSPA with or without the corresponding donor Donor HSPA , and indels were elicited and screened for resistance by puromycin.
  • the group in which no donor was added was co-transfected with a plasmid expressing a puromycin resistance gene.
  • the indels efficiency of all five genes was evaluated by the T7E1 assay (see Table 8 for the primers used), and the results are shown in Figure 8e.
  • the mutation rate at the HSPA1A site was increased by about 5.5 compared to the sgRNA HSPA alone.
  • the mutation rate at the HSPA1B site increased by about 6.1-fold
  • the mutation rate at the HSPA1L site increased by about 3.4-fold
  • the mutation rate at the HSPA6 site increased by about 6.6-fold.
  • the target region in the HSPA family gene in the mixed cells obtained by co-transfection with the donor and not co-transfected with the donor was sequenced, and the results are shown in Figure 10. The results indicate whether there is any supply at the HSPA family locus. Transfection, cell mixing and sequencing The results were consistent with the results of the T7E1 assay.
  • the T7E1 assay demonstrated that the selected population was highly enriched for cells carrying the target mutation, and the enrichment factor was approximately 753 (5.5*6.1*3.4*6.6) compared to the conventional method without donor. Considering that this calculation does not consider genes with donor insertion, the actual efficiency is even higher.
  • Example 8 Enrichment of knockout events on the CSPG4 gene in SC-8 cells using linear donor DNA containing universal sgRNA
  • sgRNA Target sequence (PAM) (5' to 3') Predicted knockout efficiency sgRNA Universal_1 GTACGGGGCGATCATCCACA (CGG) 0.982784325 sgRNA Universal_2 GCAAAAGTGGCATAAAACCG (CGG) 0.971302462 sgRNA Universal_3 TATCGCTTCCGATTAGTCCG (CGG) 0.968382667 sgRNA Universal_4 CTATCTCGAGTGGTAATGCG (CGG) 0.966411034 sgRNA Universal_5 GTAGCTGCTGTAAATCGCAT (CGG) 0.963330804 sgRNA Universal_6 TATACCAGACCACAGCGCCG (CGG) 0.962367571 sgRNA Universal_7 GCACGAGGTGAACAGCCGCT (CGG) 0.960224565
  • Ten linear donor DNAs (Donor sgRNA_Universal_1 to 10-puro ) were constructed based on the above 10 universal sgRNAs, respectively.
  • linear donor DNAs include: a 20 bp protection sequence, a sgRNA Universal_1 to 10 target sequence, a reverse stop codon, a CMV promoter-driven puromycin resistance gene, and a forward termination code from the 5' to the 3' end, respectively.
  • Child 20 bp protection sequence.
  • the two sgRNAs in tandem are sgRNA CSPG4 and sgRNA Universal_1 ⁇ 10, respectively .
  • sgRNA CSPG4 targets the receptor CSPG4 of TcdB toxin
  • sgRNA Universal_1-10 targets the target sequence in the corresponding donor DNA (Donor sgRNA_Universal_1 to 10-puro ).
  • the cell line used in the transfection experiment was a cell line stably expressing Cas9 (SC-8), and SC-8 cells were co-transfected with ten tandem plasmids Plasmid pgRNA_Universal_1-10 and corresponding donor DNA (Donor sgRNA_Universal_1-10-puro ).
  • SC-8 cells were transfected with ten linear donor DNAs (Donor sgRNA_Universal_1 to 10-puro ) alone.
  • the puromycin was added for resistance screening to obtain a pooled population, and the screening results are shown in the following table.
  • sgRNA Universal_1 , sgRNA Universal_3 , sgRNA Universal_6 and sgRNA Universal_9 have better sgRNA effects, so the subsequent experiments using the four sgRNA corresponding mixed colonies as experimental subjects.
  • TcdB toxin was added to the four mixed colonies for screening, and the cell survival was observed after 23 hours, and the results of the experiment are shown in FIG. It can be seen that after 23 hours of TcdB toxin addition, the cell viability corresponding to the sgRNA Universal_1 and sgRNA Universal_9 experimental groups was significantly higher than the other two groups, indicating that sgRNA Universal_1 and sgRNA Universal_9 have higher knockout efficiency.
  • HeLa, HeLaOC and HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM, 10-013-CV, Corning, Tewksbury, MA) supplemented with 10% fetal bovine serum (FBS, Lanzhou Braun Biotechnology Co., Ltd., Lanzhou, China) In USA, the temperature was 37 ° C and 5% CO 2 was passed. For transfection, all cells were seeded on 6-well plates and transfected with X-tremeGENE HP (06366546001, Roche, Mannheim, Germany) according to the supplier's instructions.
  • Opti-MEM I Reduced Serum Medium 31985088, Thermo Fisher Scientific, Grand Island, NY, USA. The mixture was incubated for 15 minutes at room temperature and then added to the cells.
  • the oligonucleotide of each sgRNA coding sequence was designed separately (see Table 9) and synthesized (Beijing Rui Boxing Biotechnology Co., Ltd.).
  • the oligonucleotide was dissolved to a concentration of 10 ⁇ M with 1 ⁇ TE, and the oligonucleotide was mixed with TransTaq HiFi Buffer II (K10222, Beijing Quanjin Biotechnology Co., Ltd.), heated to 95 ° C for 3 minutes, and then slowly cooled. Up to 4 ° C. These annealed oligonucleotide pairs were phosphorylated for 30 minutes at 37 ° C, and after heat inactivation, the product was ligated into the sgRNA backbone vector using the "Golden Gate" method.
  • the scaffold sequence of the gRNA and the U6 promoter were amplified with primers containing two gRNA coding sequences (Table 5), and the PCR product was then purified and ligated into the sgRNA backbone vector using the "Golden Gate” method.
  • the sgRNA backbone vector of the present invention modified the sgRNA backbone compared to the previously reported sgRNA backbone vector [17] [38] and replaced the EGFP sequence with the mCherry coding sequence.
  • Genomic DNA was extracted using a DNeasy Blood&Tissue kit (69504, Qiagen, Hilden, Germany), and the genomic region containing the gRNA target sequence was subjected to PCR amplification.
  • the primer sequences used in the assay are shown in Table 2, Table 5, Table 7, and Table 8.
  • the primer sequence used was such that 300-500 ng of PCR product was mixed with 10 x NEB Buffer 2 in a 50 ⁇ l system, and after heating at 95 ° C for 3 minutes, it was slowly cooled to room temperature. The obtained product was incubated with 0.5 ⁇ l of T7E1 for 15 min at 37 ° C for agarose gel electrophoresis.
  • the electrophoresis pattern was analyzed by Image J image analysis software to analyze the efficiency of band cleavage, indicating the efficiency of sgRNA to produce Indels.
  • the sequence containing the donor and stop codons of the CMV-driven puromycin resistance gene or EGFP gene was pre-generated and cloned into the pEASY-T5-Zero cloning vector (CT501-02, Beijing Quanjin Biotechnology Co., Ltd.) As a generic template.
  • the template was amplified using primers containing sgRNA cleavage target sites and protection sequences as shown in Table 10.
  • Cells were transfected with 1 ⁇ g of purified linear donor PCR product and 1 ⁇ g of sgRNA/pgRNA in HeLa OC cells, and treated with 1 ⁇ g/ml of puromycin two weeks after transfection.
  • Cells were transfected with 1 ⁇ g of donor and 0.5 ⁇ g of sgRNA/pgRNA and 0.5 ⁇ g of Cas9 plasmid in HeLa and HEK293T cells. Cells were then treated with 1 [mu]g/ml puromycin two weeks after transfection, or EGFP positive by fluorescence activated cell sorting (FACS), depending on which type of donor was used.
  • FACS fluorescence activated cell sorting
  • the Splinkerette PCR method has been previously reported (Potter, CJ & Luo, L. Splinkerette PCR for mapping transposable elements in Drosophila. PLoS One 5, e10168 (2010); Uren, AGet al. A high-throughput splinkerette-PCR method for the isolation And sequencing of retroviral insertion sites. Nat Protoc 4,789-798 (2009); Yin, B. & Largaespada, DAPCR-based procedures to isolate insertion sites of DNA elements. Biotechniques 43,79-84 (2007)).
  • the primers and adaptor sequences used are shown in Table 11.

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Abstract

提供一种供体构建体，以及基因敲除方法及其系统和试剂盒。所述体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA。所述基因敲除方法利用供体构建体中包含的标记物基因富集被基因敲除的细胞，提高通过序列特异性核酸酶产生基因敲除的效率。

Description

基因敲除方法 技术领域
本发明涉及基因编辑技术，具体涉及基因敲除方法。
背景技术
基因编辑技术彻底改变了对基因功能的实验研究。三种主要的技术，ZFN(锌指核酸酶)[1]，TALENs(转录激活物样效应子核酸酶)[2-4]和CRISPR/Cas9系统[5-7]，使用不同的机制产生序列特异性双链断裂(double-strand breaks,DSBs)并随后引发天然修复系统完成序列特异性修饰[8,9]。这些技术在功能基因研究[10]、染色体位点的动态和实时成像[11,12]、疾病突变的校正[13]、基因治疗[14]等方面具有广泛的应用。CRISPR/Cas9系统因其高效性和易于操作，变得特别受欢迎。CRISPR/Cas9系统原本被细菌免疫系统用来抵御外源病毒或质粒，在II类CRISPR系统中，Cas9核酸内切酶在sgRNA的引导下切割双链DNA，造成基因组双链断裂，利用细胞基因组修复的不稳定性产生修复错误(碱基的缺失或插入)，从而产生基因编辑的效果。
虽然CRISPR/Cas9系统在基于设计的和序列特异性的基因组研究中具有前所有未有的优势，但CRISPR/Cas9系统引发的基因编辑在细胞群体中仍然属于稀有事件，要得到真正的基因被编辑的单克隆，需要进行繁琐的劳动，因此该系统在技术上仍然具有挑战性，即使是对于在哺乳动物细胞中产生基因敲除这样的简单任务来说[15]。已进行了各种努力来提高产生基因敲除的方案的效率，例如整合CRISPR/Cas9系统以永久表达Cas9和sgRNA[16]、预先产生稳定表达Cas9的细胞系[17]、增强非同源末端连接(NHEJ)途径[18]、通过同时破坏能实现特异性药物选择的单独基因来富集基因-靶向事件[19]、以及使用代理报告子(surrogate reporter)富集基因敲除[20,21]。但是，各种缺点限制了这些技术的广泛应用。特别是，在哺乳动物细胞中产生多基因敲除仍然是难以完成的任务。当使用传统方法时，即使是敲除单个基因有时也是长时间的，繁重的和高风险的任务[22]，因为它们缺乏对含有靶基因修饰的稀少克隆的有效富集。
如果靶基因的破坏可以导致能够用于富集的表型变化，可以容易地获得基因敲除克隆， 例如Hela CSPG4-/-细胞赋予了对艰难梭菌(Clostridium difficile)毒素B的抗性[23]。但是，该策略无法通用。传统方法是要共转染表达抗生素抗性或荧光蛋白的质粒[23,24]，但是这种方法不会富集含有靶向修饰的少量细胞。
已报道，外源dsDNA片段可能通过不同的修复机制被整合到具有DSBs的染色体位点上。通过同源重组(HR)修复需要构建长侧翼序列，整合效率较低，而通过非同源末端连接(Non-Homologous end joing,NHEJ)DNA修复的整合效率[27,28]通常高于同源重组修复[29]。已有研究使用CRISPR/Cas引发的NHEJ介导外源线性供体DNA的插入，以达到基因敲入的目的[30-34]。
发明内容
本发明提供供体构建体，以及基因敲除方法及其系统和试剂盒。本发明的基因敲除方法利用所述供体构建体中包含的标记物基因富集被基因敲除的细胞，提高通过序列特异性核酸酶产生基因敲除的效率。
根据本发明的一个方面，提供供体构建体，所述供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的靶序列，所述靶序列含有可被所述序列特异性核酸酶切割的靶位点；位于两端的保护序列；其中所述表达盒包含由启动子驱动的标记物基因。
在本发明中，所述线性供体DNA是双链线性供体DNA。
在优选的实施方案中，所述供体构建体是线性供体DNA。
在一些实施方案中，所述序列特异性核酸酶是锌指核酸酶(ZFN)。
在另一些实施方案中，所述序列特异性核酸酶是转录激活物样效应子核酸酶(TALEN)。
在另一些实施方案中，所述序列特异性核酸酶是Cas9核酸酶。
在另一些实施方案中，所述序列特异性核酸酶是NgAgo核酸酶。
在一些实施方案中，所述线性供体DNA仅在5'端或3'端具有靶序列。
在一些实施方案中，所述线性供体DNA在两端分别具有靶序列。
在一些实施方案中，所述线性供体DNA中两端的靶序列相同。
在一些实施方案中，所述线性供体DNA中两端的靶序列不同。在进一步的实施方案中，所述线性供体DNA中两端的不同靶序列来自于相同的基因。在其它进一步的实施方案中，所 述线性供体DNA中两端的不同靶序列来自于不同的基因。
在优选的实施方案中，所述标记物基因是抗生素抗性基因或荧光蛋白基因。
在优选的实施方案中，保护序列的长度为5-30bp，最优选20bp。
根据本发明的另一个方面，提供在细胞中产生基因敲除的方法，所述方法包括以下步骤：
(1)向细胞中引入能切割细胞基因组中特定靶位点的序列特异性核酸酶和供体构建体；
其中所述供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的靶序列，所述靶序列含有可被所述序列特异性核酸酶切割的靶位点；位于两端的保护序列；所述表达盒包含由启动子驱动的标记物基因；
其中线性供体DNA通过非同源末端连接被插入到细胞基因组中的特定靶位点；
(2)筛选所述标记物表达阳性的细胞。
在本发明中，所述线性供体DNA是双链线性供体DNA。
在优选的实施方案中，所述供体构建体是线性供体DNA。
在一些实施方案中，所述线性供体DNA仅在5'端或3'端具有靶序列。
在一些实施方案中，所述线性供体DNA在两端分别具有靶序列。
在一些实施方案中，所述线性供体DNA中两端的靶序列相同。
在一些实施方案中，所述线性供体DNA中两端的靶序列不同。在进一步的实施方案中，所述线性供体DNA中两端的不同靶序列来自于相同的基因。在其它进一步的实施方案中，所述线性供体DNA中两端的不同靶序列来自于不同的基因。
在一些实施方案中，所述序列特异性核酸酶是锌指核酸酶(ZFN)。
在另一些实施方案中，所述序列特异性核酸酶是转录激活物样效应子核酸酶(TALEN)。
在另一些实施方案中，所述序列特异性核酸酶是Cas9核酸酶。
在优选的实施方案中，所述方法还包括向细胞中引入识别细胞基因组中特定靶位点的向导RNA(gRNA)，线性供体DNA中的靶序列包含被所述gRNA识别的靶位点。
在一些实施方案中，所述gRNA是sgRNA。
在更优选的实施方案中，所述方法还包括向细胞中引入识别细胞基因组中单个特定靶位点的sgRNA，包含被所述sgRNA识别的靶位点的靶序列位于线性供体DNA的5'端和/或3'端。在一些实施方案中，包含被所述sgRNA识别的靶位点的靶序列来自于细胞基因组中的单个基 因。在一些实施方案中，包含被所述sgRNA识别的靶位点的靶序列是细胞基因组中两个或多个基因的共有序列，条件是所述共有序列与所述两个或多个基因的任何一个上与共有序列对应位置处的序列具有不超过一个碱基的差异。
在另一些更优选的实施方案中，所述方法还包括向细胞中引入识别细胞基因组中一个基因上的两个特定靶位点的两个sgRNA，分别包含被所述两个sgRNA识别的两个靶位点的两个靶序列分别位于两个线性供体DNA中，或者分别位于同一个线性供体DNA的两端。
在另一些更优选的实施方案中，所述方法还包括向细胞中引入识别细胞基因组中两个或更多个特定靶位点的两个或多个sgRNA，分别包含被所述两个或多个sgRNA识别的两个或更多个靶位点的两个或更多个靶序列分别位于同一个线性供体DNA的两端或者位于不同的线性供体DNA中。其中所述细胞基因组中两个或更多个特定靶位点分别位于不同的基因上。
在另一些实施方案中，所述序列特异性核酸酶是NgAgo核酸酶。
在优选的实施方案中，所述方法还包括向细胞中引入引入识别细胞基因组中特定靶位点的向导DNA(gDNA)，其中线性供体DNA中的靶序列包含被所述gDNA识别的靶位点。
本发明中，所述基因敲除可以是单个基因敲除或多基因敲除。所述多基因敲除是两个基因或更多个基因的敲除，例如三个、四个、五个或更多个基因的敲除。
在优选的实施方案中，所述标记物基因是抗生素抗性基因或荧光蛋白基因。
在一个优选的实施方案中，通过药物抗性筛选细胞。
在另一个优选的实施方案中，通过FACS方法筛选细胞。
在优选的实施方案中，保护序列的长度为5-30bp，最优选20bp。
根据本发明的另一个方面，提供用于基因敲除的系统或试剂盒，其中包括：能切割细胞基因组中特定靶位点的序列特异性核酸酶和供体构建体；
其中所述供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的靶序列，所述靶序列含有可被所述序列特异性核酸酶切割的靶位点；位于两端的保护序列；所述表达盒包含由启动子驱动的标记物基因。
本发明中，线性供体DNA是双链线性供体DNA。
在一些实施方案中，所述供体构建体是线性供体DNA。在另一些实施方案中，所述供体构建体是环形供体构建体且可在细胞中被切割产生线性供体DNA。
在一些实施方案中，所述序列特异性核酸酶是锌指核酸酶(ZFN)。
在另一些实施方案中，所述序列特异性核酸酶是转录激活物样效应子核酸酶(TALEN)。
在另一些实施方案中，所述序列特异性核酸酶是Cas9核酸酶。
在优选的实施方案中，所述系统或试剂盒还包括识别细胞基因组中特定靶位点的sgRNA，其中线性供体DNA中的靶序列包含被所述sgRNA识别的靶位点。
在一些实施方案中，所述gRNA是sgRNA。
在另一些实施方案中，所述序列特异性核酸酶是NgAgo核酸酶。
在优选的实施方案中，所述系统或试剂盒还包括识别细胞基因组中特定靶位点的gDNA，其中线性供体DNA中的靶序列包含被所述gDNA识别的靶位点。
在优选的实施方案中，所述标记物基因是抗生素抗性基因或荧光蛋白基因。
在优选的实施方案中，保护序列的长度为5-30bp，最优选20bp。
本发明中，所述切割是产生双链断裂(DSBs)。
根据本发明的另一个方面，提供通用供体构建体，所述通用供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的通用靶序列，所述通用靶序列含有可被Cas9核酸酶切割的靶位点；位于两端的保护序列；
其中所述表达盒包含由启动子驱动的标记物基因；
其中所述通用靶序列在待进行基因敲除的细胞的基因组中不存在。
在一些实施方案中，所述通用供体构建体是线性供体DNA。
在一些实施方案中，所述线性供体DNA是双链线性供体DNA。
在一些实施方案中，所述线性供体DNA仅在5'端或3'端具有所述通用靶序列。
在一些实施方案中，所述线性供体DNA在两端分别具有所述通用靶序列。
在优选的实施方案中，所述标记物基因是抗生素抗性基因或荧光蛋白基因。
在优选的实施方案中，保护序列的长度为5-30bp，最优选20bp。
在优选的实施方案中，所述通用供体构建体中的通用靶序列含有5'-GTACGGGGCGATCATCCACA-3'或5'-AATCGACTCGAACTTCGTGT-3'。
根据本发明的另一个方面，提供在细胞中产生基因敲除的方法，所述方法包括以下步骤：
(1)向细胞中引入：
(a)Cas9核酸酶；
(b)识别细胞基因组中特定靶序列的gRNA；
(c)通用供体构建体，其中所述通用供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的通用靶序列，所述通用靶序列含有可被Cas9核酸酶切割的靶位点；位于两端的保护序列；
其中所述表达盒包含由启动子驱动的标记物基因；
其中所述通用靶序列在待进行基因敲除的细胞的基因组中不存在；
(d)识别线性供体DNA中包含的通用靶序列的gRNA。
(2)所述线性供体DNA通过非同源末端连接被插入到细胞基因组中的特定靶位点；
(3)筛选所述标记物表达阳性的细胞。
在一些实施方案中，所述供体构建体是线性供体DNA。
在一些实施方案中，所述线性供体DNA是双链线性供体DNA。
在一些实施方案中，所述线性供体DNA仅在5'端或3'端具有所述通用靶序列。
在一些实施方案中，所述线性供体DNA在两端均具有所述通用靶序列。
在一些实施方案中，所述识别细胞基因组中特定靶序列的gRNA可以是一种gRNA，或者多种识别细胞基因组中不同靶序列的gRNA，例如2种、3种或更多种识别细胞基因组中不同靶序列的gRNA。所述不同靶序列可以位于同一个基因中，或者可以位于不同的基因中。当所述不同靶序列分别位于不同的基因中时，可以实现多个基因的敲除。
因此，本发明中，所述基因敲除可以是单个基因敲除或多基因敲除。所述多基因敲除是两个基因或更多个基因的敲除，例如三个、四个、五个或更多个基因的敲除。
在一些实施方案中，所述识别细胞基因组中特定靶序列的gRNA是sgRNA。
在一些实施方案中，所述识别线性供体DNA中包含的通用靶序列的gRNA是sgRNA。
在一些实施方案中，所述识别细胞基因组中特定靶序列的sgRNA和所述识别线性供体DNA中包含的通用靶序列的sgRNA位于相同的载体中。
在一些实施方案中，所述识别细胞基因组中特定靶序列的sgRNA和所述识别线性供体DNA中包含的通用靶序列的sgRNA位于不同的载体中。
在优选的实施方案中，所述标记物基因是抗生素抗性基因或荧光蛋白基因。
在一个优选的实施方案中，通过药物抗性筛选细胞。
在另一个优选的实施方案中，通过FACS方法筛选细胞。
在优选的实施方案中，保护序列的长度为5-30bp，最优选20bp。
在优选的实施方案中，所述通用供体构建体中的通用靶序列含有5'-GTACGGGGCGATCATCCACA-3'或5'-AATCGACTCGAACTTCGTGT-3'。
根据本发明的另一个方面，提供用于基因敲除的系统或试剂盒，其中包括：
(1)Cas9核酸酶或能够表达Cas9核酸酶的载体或细胞；
(2)识别细胞基因组中特定靶序列的gRNA；
(3)通用供体构建体，其中所述通用供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的通用靶序列，所述通用靶序列含有可被Cas9核酸酶切割的靶位点；位于两端的保护序列；
其中所述表达盒包含由启动子驱动的标记物基因；
其中所述通用靶序列在待进行基因敲除的细胞的基因组中不存在；
(4)识别线性供体DNA中包含的通用靶序列的gRNA。
在一些实施方案中，线性供体DNA是双链线性供体DNA。
在一些实施方案中，所述供体构建体是线性供体DNA。
在另一些实施方案中，所述供体构建体是环形供体构建体且可在细胞中被切割产生线性供体DNA。
在一些实施方案中，所述识别细胞基因组中特定靶序列的gRNA可以是一种gRNA，或者多种识别细胞基因组中不同靶序列的gRNA，例如2种、3种或更多种识别细胞基因组中不同靶序列的gRNA。所述不同靶序列可以位于同一个基因中，或者可以位于不同的基因中。当所述不同靶序列分别位于不同的基因中时，可以实现多个基因的敲除。
在一些实施方案中，所述识别细胞基因组中特定靶序列的gRNA是sgRNA。
在一些实施方案中，所述识别线性供体DNA中包含的通用靶序列的gRNA是sgRNA。
在一些实施方案中，所述识别细胞基因组中特定靶序列的gRNA和所述识别线性供体DNA中包含的通用靶序列的gRNA位于相同的载体中。
在一些实施方案中，所述识别细胞基因组中特定靶序列的gRNA和所述识别线性供体 DNA中包含的通用靶序列的gRNA位于不同的载体中。
在优选的实施方案中，所述标记物基因是抗生素抗性基因或荧光蛋白基因。
在优选的实施方案中，保护序列的长度为5-30bp，最优选20bp。
本发明中，所述切割是产生双链断裂(DSBs)。
在优选的实施方案中，所述通用供体构建体中的靶序列含有5'-GTACGGGGCGATCATCCACA-3'或5'-AATCGACTCGAACTTCGTGT-3'。
本发明可以通过将标记物基因插入到基因敲除的切割靶位点上，通过标记物有效富集具有产生基因敲除的稀少克隆。本发明对于sgRNAs设计困难的基因的靶向，以及在需要同时靶向几个基因敲除的情况下，是特别有用的。该方法有助于各种产生DNA双链断裂的基因编辑系统，特别是CRISPR系统在基因及其功能的生物医学领域的更广泛应用。
附图说明
图1是通过嘌呤霉素选择来富集在HeLa细胞中的ANTXR1基因上含有Cas9/gRNA靶向突变的细胞的供体设计和实验验证。(a)线性供体在ANTXR1基因上的sgRNA-或pgRNA靶向位点的基于NHEJ的敲入的示意图，基因组位点和线性供体上具有gRNA的切割位点sg1和sg2，终止密码子用***标记，箭头指向阅读框的方向。(b)用供体(加gRNA或不加gRNA)转染的细胞的嘌呤霉素抗性克隆的MTT染色。(c)用sgRNA/pgRNA(加(深色条)或不加(浅色条)它们相应的供体)转染的HeLa细胞的ANTXR1敲除率比较，ANTXR1敲除率表示为具有PA/LFnDTA抗性的细胞的百分比。在进行PA/LFnDTA抗性分析之前用嘌呤霉素(1μg/ml)选择细胞。误差线表示s.d.(n＝3)，t-test，**P<0.01，***P<0.001。(d)使用不同的gRNAs及其供体富集ANTXR1敲除细胞的总结。
图2是通过供体介导的嘌呤霉素抗性选择在混合群落和单克隆中富集ANTXR1敲除细胞的实验验证。(a)不同HeLa细胞组使用或不使用PA/LFnDTA处理的图像。用sgRNA或pgRNA加或不加它们相应的线性供体转染获得混合细胞，比例尺为200μm。(b)对具有嘌呤霉素抗性(puro+)单克隆的线性供体整合的ANTXR1位点的PCR验证，克隆从sgRNA2ANTXR1/DonorANTXR1-sg2(左)或pgRNAANTXR1/DonorANTXR1-pg(右)转染的HeLa细胞获得。
图3是通过嘌呤霉素选择富集HeLa细胞中HEBGF破坏事件的供体设计和实验验证。(a)靶向HBEGF基因的供体设计。(b)用线性供体DonorHBEGF-sg1(加或不加Cas9/sgRNA)转染的 细胞中嘌呤霉素抗性克隆的MTT染色。(c)不同HeLa细胞组使用或不使用DT(40ng/ml)处理的图像。用sgRNA(sgRNA1HBEGF)(加或不加它的相应线性供体(DonorHBEGF-sg1))转染获得混合细胞，比例尺为200μm。(d)用sgRNA(sgRNA1HBEGF)、表达Cas9的质粒和含有嘌呤霉素抗性基因(浅色条)的报告质粒，或者用sgRNA、表达Cas9的质粒和线性供体(DonorHBEGF-sg1)(深色条)转染的HeLa细胞的HBEGF敲除率，HBEGF敲除率表示为对具有DT抗性的细胞的百分比。在分析DT抗性之前用嘌呤霉素(1μg/ml)选择细胞。误差线表示s.d.(n＝3)，t-test，***P<0.001。
图4是通过EGFP富集HEK293T细胞中HBEGF破坏事件的供体设计和实验验证。(a)靶向HBEGF基因的供体设计。(b)不同HEK293T细胞组使用或不使用DT(40ng/ml)处理的的图像。用sgRNA(sgRNA2HBEGF)(加或不加它们相应的线性供体(DonorHBEGF-sg2))转染获得混合细胞，比例尺200μm。(c)用表达mCherry的sgRNA(sgRNA2HBEGF)质粒和表达Cas9的质粒(浅色条)，或者用sgRNA、表达Cas9的质粒和线性供体(DonorHBEGF-sg2，EGFP)(深色条)转染的HEK293T细胞的HBEGF敲除率，HBEGF敲除率用对DT具有抗性的细胞的百分比表示。在分析DT抗性之前用FACS选择细胞。误差线表示s.d.(n＝3)，t-test，***P<0.05。
图5是通过嘌呤霉素选择富集HeLaOC细胞中ANTXR1破坏事件的供体设计和实验验证。(a)靶向ANTXR1的供体设计。供体在5'端含有一个sgRNA切割位点(DonorANTXR1-sg1或DonorANTXR1-sg2)或在两端含有两个gRNAs(DonorANTXR1-pg)。(b)是用供体(加gRNA或不加gRNA)转染的细胞中嘌呤霉素抗性克隆的MTT染色。(c)不同HeLaOC细胞组使用或不使用PA/LFnDTA处理的图像。用sgRNA或pgRNA加或不加它的相应线性供体转染获得混合细胞，比例尺为200μm。(d)用sgRNAs加或不加它们的相应供体转染的HeLaOC细胞的ANTXR1敲除率，ANTXR1敲除率表示为为具有PA/LFnDTA抗性的细胞的百分比。在分析PA/LFnDTA抗性之前用嘌呤霉素(1μg/ml)选择细胞。误差线表示s.d.(n＝3)，t-test，***P<0.001。(e)对嘌呤霉素抗性单克隆的线性供体整合的ANTXR1位点的PCR验证。(f)使用不同的gRNAs和它们的供体富集ANTXR1敲除细胞的总结。
图6是在HeLaOC细胞中通过splinkerette PCR(spPCR)分析对供体插入进行脱靶评估。(a)用于spPCR分析的接头(adaptor)和引物的设计。Splink1和Splink2引物与接头序列匹配，引物R1和R2与线性供体序列匹配。(b)spPCR反应结果。
图7是通过嘌呤霉素选择富集HeLaOC细胞中的HBEGF破坏事件的供体设计和实验验证。(a)靶向HBEGF的供体设计。供体在5'端含有一个sgRNA切割位点(DonorHBEGF-sg1或 DonorHBEGF-sg2)或在两端含有两个gRNAs(DonorHBEGF-pg)。(b)用供体(加或不加sgRNA/pgRNA)转染的细胞中嘌呤霉素抗性克隆的MTT染色。(c)对嘌呤霉素抗性单克隆的线性供体整合的HBEGF位点的PCR验证。(d)使用不同的gRNAs和它们的供体的HBEGF敲除细胞富集的总结。
图8是在HeLaOC细胞中一步产生两个或多个基因敲除的供体设计和实验验证。(a)线性供体在PSEN1和PSEN2基因上的sgRNA-或pgRNA靶向位点的基于NHEJ的敲入的示意图。(b-c)基因组中PSEN1和PSEN2的部分编码序列，含有sgRNA编码区域(加下划线)和突变等位基因的测序分析。克隆1(b)来自于用pgRNAPSEN1+PSEN2/DonorPSEN1+PSEN2转染的HeLaOC细胞。克隆2(c)来自于用pgRNAPSEN1+PSEN2/DonorPSEN1+DonorPSEN2转染的HeLaOC细胞。阴影区域的核苷酸代表引导Cas9进行DNA识别和切割的PAM序列。虚线表示缺失，高字母表示核苷酸插入，背景中的浅灰色箭头表示供体中CMV启动子的方向。(d)HSPA基因家族的多序列比对分析，显示其共有序列，靶向五个HSPA家族基因的共有序列的sgRNA，以及用于富集含有多基因突变的细胞的通用线性供体(DonorHSPA)的设计。黑色阴影核苷酸代表所有五个HSPA基因的共有序列。深灰色阴影核苷酸代表三个或四个HSPA基因的共有序列，浅灰色阴影核苷酸代表非共有核苷酸。(e)在缺少和存在DonorHSPA的条件下，在进行了嘌呤霉素选择后，sgRNAHSPA在五个靶基因上引发的indels。误差线表示s.d.(n＝3)，t-test，**P<0.01，***P<0.001。(f)含有sgRNA靶向区域(加下划线)的HeLa克隆3的基因组中HSPA1A,HSPA1B,HSPA1L和HSPA6基因的部分编码序列。克隆3来自于用sgRNAHSPA/DonorHSPA转染的HeLaOC细胞。阴影部分的核苷酸代表PAM序列，虚线代表缺失。背景中的浅灰色箭头表示供体中的CMV启动子方向。
图9是HeLaOC细胞中PSEN1和PSEN2sgRNA效率评价和单个克隆识别。(a)通过T7E1分析对在PSEN1和PSEN2位点对sgRNAPSEN1、sgRNAPSEN2和pgRNAPSEN导致的indels效率进行评价。误差线表示s.d.(n＝3)。(b)用供体(加或不加pgRNAPSEN)转染的细胞中嘌呤霉素抗性克隆的MTT染色。(c)嘌呤霉素抗性单克隆的线性供体整合的PSEN1(L3/R3)和PSEN2(L4/R4)两个位点的PCR结果。
图10是用或不用供体转染的混合细胞中HSPA家族基因的靶区域的测序图。sgRNA靶位点用阴影标出，这些测序分析中不包括含有供体插入的靶区域。
图11是五个HSPA家族基因，HSPA1A、HSPA1B、HSPA1L、HSPA6和HSPA2的靶位点上的供体插入的单克隆鉴别。(a)嘌呤霉素抗性单克隆在所有五个基因位点上的线性供体整合的 PCR验证结果。(b)四个基因位点HSPA1A、HSPA1B、HSPA1L、HSPA6中供体插入结果的总结。
图12是利用包含通用sgRNA的供体进行基因敲除的实验流程图。
图13是利用包含通用sgRNA的供体进行基因敲除的基因敲除效率验证。
具体实施方式
本发明提供了新的供体构建体和基因敲除方法，该方法利用线性供体DNA提高通过序列特异性核酸酶产生基因敲除的效率。本发明的线性供体DNA上包含至少一个可以被序列特异性核酸酶切割的靶位点。根据细胞基因组中的靶位点设计线性供体DNA中包含的靶位点，使得能够切割细胞基因组中靶位点的序列特异性核酸酶也能切割线性供体DNA中包含的靶位点。将序列特异性核酸酶和供体构建体引入细胞后，序列特异性核酸酶在细胞中特定靶位点产生双链断裂(DSBs)时，会同时切割线性供体DNA中包含的至少一个靶位点，由此可以使得线性供体DNA以较高的效率通过非同源末端连接(Non-Homologous end joining,NHEJ)途径被插入到细胞基因组中被切割的靶位点上，随后通过该标记物对细胞进行选择，可以有效地富集因在基因组的特定靶位点被切割而产生基因敲除的细胞，极大地提高通过序列特异性核酸酶产生基因敲除的效率。
根据细胞基因组中的靶位点设计线性供体DNA中包含的靶位点，所获得的线性供体DNA是特异性线性供体，当需要在细胞基因组中的不同靶位点进行基因敲除时，需要根据该靶位点的序列构建配套使用的线性供体DNA。因此，为了进一步优化本发明，发明人在本发明中进一步提供通用线性供体DNA，所述通用线性供体DNA上包含可以被序列特异性核酸酶切割的通用靶序列，该通用靶序列在待进行基因敲除的细胞的基因组中不存在，即在待进行基因敲除的细胞的基因组中不存在与所述通用靶序列相同的可被所述序列特异性核酸酶切割的序列。在此情况下，将序列特异性核酸酶和通用线性供体DNA引入细胞后，序列特异性核酸酶在细胞中特定靶位点产生双链断裂(DSBs)时，通用线性供体DNA中包含的通用靶序列也通过识别该靶序列的通用gRNA被所述序列特异性核酸酶切割，此时线性供体DNA仍然可以较高的效率通过非同源末端连接(Non-Homologous end joining,NHEJ)途径被插入到细胞基因组中被切割的靶位点上，随后通过该标记物对细胞进行选择，可以有效地富集因在基因组的特定靶位点被切割而产生基因敲除的细胞，同样可以极大地提高通过序列特异性核酸酶产生基因敲除的效率。所述通用线性供体DNA中的靶序列与待敲除基因无关，它可以作为通用的 供体用于不同细胞中的不同目标基因的敲除，均可以提高通过序列特异性核酸酶产生基因敲除的效率。通用线性供体DNA特别可用于使用Cas9/CRISPR系统进行基因敲除的情况，该系统使用gRNA(优选sgRNA)靶向目标序列，当进行基因敲除时，只需构建针对细胞基因组中特定靶位点的gRNA，无需专门构建配套使用的线性供体DNA，直接使用通用线性供体DNA和靶向该通用线性供体DNA的gRNA即可，由此降低了操作复杂度，提高了效率。
已有报道，如果同源等位基因中的一个被修饰，则靶等位基因的突变频率通常较高[25,26]。因此，虽然不希望受到理论的限制，但发明人推测，如果能够在靶等位基因的一个上的特定位点插入供体，并选择表达供体含有的标记物基因的克隆，可能能够富集那些所有等位基因都被修饰的稀有事件。
本发明中，“基因敲除”是通过基因编辑实现基因功能的丧失。通常所追求的基因敲除效果是两个等位基因同时敲除，此时对应蛋白丧失功能，得到基因敲除细胞系。如果只有一个等位基因被敲除，则蛋白还能发挥部分作用，仅仅是蛋白功能下调。利用本发明的线性供体DNA和本发明的方法，可以有效富集两个等位基因都被敲除的细胞。
本发明的供体构建体是双链DNA。本发明的供体构建体本身即可以是线性供体DNA。或者本发明的供体构建体可以是包含线性供体DNA的环形DNA分子，当被引入到细胞中时，在细胞中被切割产生线性供体DNA。在细胞中切割环形供体构建体产生线性供体DNA的方法是本领域熟知的。例如，环形构建体中可以在线性供体DNA的5'端上游和3'端下游进一步包含另一种序列特异性核酸酶的切割位点。
在本发明的方法中，可以进一步包括向细胞引入另一种序列特异性核酸酶，该序列特异性核酸酶在细胞中切割环形构建体中线性供体DNA的5'端上游和3'端下游的序列，由此产生线性供体DNA。
本发明的线性供体DNA中，“反向终止密码子”是指该密码子的方向与表达盒的阅读框的方向相反。“正向终止密码子”是指该密码子的方向与表达盒的阅读框的方向相同。终止密码子的作用是：无论线性供体被正向或者反向插入基因组，两个三联终止密码子都能中止内源及外源基因表达。
本发明的线性供体DNA中“保护序列”可以是任意序列，优选保护序列与同一线性供体DNA中的靶序列不同。保护序列的长度可以是5-30bp，优选20bp。保护序列的作用是保护线性供体DNA中的靶序列不被细胞中的酶(例如核酸外切酶)切割。
本文中所述的“标记物基因”是指其表达可以被选择或富集的任何标记物基因，即当该 标记物基因在细胞中表达时，可以通过一定方式选择和富集表达该标记物基因的细胞。可用于本发明的标记物基因包括但不限于在表达后可以用FACS分选的荧光蛋白基因，或者可以利用抗生素进行筛选的抗性基因，或者表达后可以被对应抗体识别并通过免疫染色或磁珠吸附进行筛选的蛋白基因。可用于本发明的抗性基因包括但不限于针对杀稻瘟菌素(Blasticidin)、遗传霉素(Geneticin，G-418)、潮霉素(Hygromycin B)、霉酚酸(Mycophenolic Acid)、嘌呤霉素(Puromycin)、博莱霉素(Zeocin)或新霉素(Neomycin)的抗性基因。可用于本发明的荧光蛋白基因包括但不限于蓝色荧光蛋白(Cyan Fluorescent Protein)、绿色荧光蛋白(Green Fluorescent Protein)、黄色荧光蛋白(Yellow Fluorescent Protein)、橙色荧光蛋白(Orange Fluorescent Protein)、红色荧光蛋白(Red Fluorescent Protein)、远红色荧光蛋白(Far-Red Fluorescent Protein)或可切换荧光蛋白(Switchable Fluorescent Proteins)的基因。
序列特异性核酸酶的实例包括锌指核酸酶(ZFN)。锌指核酸酶是非自然存在的，人工改造的核酸内切酶，由锌指蛋白结构域和非特异性核酸内切酶结构域组成。锌指蛋白结构域由一系列Cys2-His2锌指蛋白串联组成，每个锌指蛋白识别并结合3′到5′方向DNA链上一个特异的三联体碱基以及5′到3′方向的一个碱基。多个锌指蛋白可以串联起来形成一个锌指蛋白组，识别一段特异的碱基序列，具有很强的特异性。与锌指蛋白组相连的非特异性核酸内切酶来自FokI羧基端96个氨基酸残基组成的DNA剪切域。每个FokI单体与一个锌指蛋白组相连构成一个ZFN，识别特定的位点，当两个识别位点相距恰当的距离时(6～8bp)，两个单体ZFN相互作用产生酶切功能，从而达到DNA定点剪切的目的。针对靶序列设计8～10个锌指结构域，将这些锌结构域连在DNA核酸酶上，便可实现靶序列的双链断裂(Double strand breaks,DSBs)，进而诱发DSBs修复机制，对基因组中的特定位点进行定向改造。
序列特异性核酸酶的另一实例包括转录激活物样效应子核酸酶(TALEN)。转录激活物样效应子核酸酶主要由Fok I内切酶结构域和TALE蛋白的DNA结合结构域组合而成。TALE蛋白含有多个33-35个氨基酸组成的重复肽段，而每一个肽段都能够识别一个碱基。与ZFN一样，TALEN也能使DNA靶序列断裂，形成DSBs，进而激活DNA损伤修复机制，对基因组进行定点改造。
可用于本发明的序列特异性核酸酶系统的另一种实例包括Cas9/CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats，成簇的规律间隔的短回文重复序列)系统。Cas9/CRISPR系统利用RNA指导的DNA结合对靶DNA进行序列特异性切割，由crRNA(CRISPR-derived RNA)通过碱基配对与tracrRNA(trans-activating RNA)结合形成 tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白在与crRNA配对的靶序列上的特定位置处剪切双链DNA。与crRNA配对的靶序列通常是位于基因组PAM(原间隔区邻近基序)位点(NNG)上游的约20个核苷酸的序列。
Cas9蛋白对靶位点的切割需要借助向导RNA。术语“向导RNA”又称gRNA(guide RNA)，gRNA通常包括crRNA上与靶序列互补的核苷酸和由crRNA与tracrRNA碱基配对形成的RNA支架(Scaffold)，能够识别与crRNA配对的靶序列。gRNA可以与Cas9蛋白形成复合体并将Cas9蛋白带至靶序列并切割其中的靶位点。
gRNA通常以sgRNA(single guide RNA)的形式使用。sgRNA又称“单链向导RNA”，是crRNA和trancrRNA融合而成的一条RNA链。
可用于本发明的序列特异性核酸酶系统的另一种实例包括NgAgo核酸酶及其gDNA。NgAgo核酸酶可以与5'端磷酸化的单链向导DNA(gDNA)为结合，对与该gDNA互补的靶序列进行切割，造成DNA双链断裂。
本发明的线性供体DNA可以仅在一端具有靶序列，也可以在两端分别具有靶序列。线性供体DNA两端的靶序列可以不同。当需要在细胞基因组中两个不同靶位点上进行切割而产生基因敲除时，可以提供两个线性供体DNA，每个线性供体DNA分别包含一个相应的靶序列，也可以提供一个线性供体DNA，该线性供体DNA的每一端包含一个相应的靶序列。当需要在细胞基因组中多个不同靶位点上进行切割而产生基因敲除时，可以提供适当数量的线性供体DNA，每个线性供体DNA的一端或两端分别包含多个不同相应靶序列中的一个。例如可以提供与靶位点数量相同的线性供体DNA，每个线性供体DNA分别包含一个相应的靶序列。或者可以提供少于靶位点数量的线性供体DNA，其中全部或部分线性供体DNA的两端分别包含多个不同相应靶序列中的一个，其它线性供体DNA每个分别包含其它相应靶序列中的一个。
对于含有通用靶序列的通用线性供体DNA，则可以在其任何一端或两端具有含有所述通用靶序列，这种通用线性供体DNA的靶序列不依赖于细胞基因组中待切除的靶位点，因而可以普遍性地适用于对细胞基因组中的任何一个靶位点、任何两个靶位点、或任何更多个靶位点进行切割而产生基因敲除的情况，。
本发明所述“通用靶序列”是指可被序列特异性核酸酶切割的序列，但所述通用靶序列在待进行基因敲除的细胞的基因组中不存在，也就是说，在待进行基因敲除的细胞的基因组中不存在与所述通用靶序列相同的可被所述序列特异性核酸酶切割的序列，通用靶序列与细胞基因组上存在的可被同一序列特异性核酸酶切割的靶序列均不同。含有该通用靶位点的线 性供体DNA对于细胞基因组上的靶位点都没有特异性，因此可以普遍地适用于对细胞中任何基因的基因敲除中，无需针对需要敲除的基因即其上的靶位点构建特定的线性供体DNA。
序列特异性核酸酶可以以蛋白质的形式或者以其编码核酸序列(例如mRNA或cDNA)的形式被引入细胞。编码序列特异性核酸酶的核酸可以被包含在质粒或病毒载体中被引入细胞，例如通过转染被引入细胞。编码序列特异性核酸酶的核酸也可以通过电穿孔、脂质体、显微注射等方式被直接递送到细胞中。
供体构建体可以通过任何适合于将核酸引入细胞中的方法递送，例如通过转染被引入细胞中。
在使用Cas9/CRISPR系统和NgAgo核酸酶产生基因敲除的情况下，还要将sgRNA或gDNA引入细胞。可以通过任何适合于将RNA或DNA引入细胞中的方法递送sgRNA或gDNA。sgRNA可以以分离的RNA的形式被引入细胞。可以使用本领域中已知的任何体外转录系统通过体外转录来制备分离的sgRNA。还可以通过包含编码sgRNA的序列和启动子的载体将sgRNA引入细胞。所述载体可以是病毒载体或质粒。引入细胞的方式可以是转染。
可以向细胞中引入两个或更多个分别针对不同靶位点的sgRNA，以引导Cas9切割细胞基因组中两个或更多个不同靶位点上进行切割而产生基因敲除。所述两个或更多个sgRNA可以被包含在不同的载体中，也可以被包含在同一个载体中，例如包含一对gRNA(paired gRNA)的载体，或者包含更多个sgRNA的载体。
在本发明的方法中，当向细胞中引入两个或更多个分别针对不同靶位点的sgRNA时，同时引入包含被这些sgRNA识别的靶序列的线性供体DNA。由于线性供体DNA可以仅在5'端或3'端含有靶序列，也可以在两端分别含有靶序列，sgRNA的数量和线性供体DNA的数量可以不同，可以是一个sgRNA对应一个线性供体DNA，也可以是两个sgRNA对应两个线性供体DNA。
当使用Cas9/CRISPR系统和本发明的通用线性供体DNA进行基因敲除时，除了将通用线性供体DNA和Cas9核酸酶引入细胞，还要向细胞中引入针对细胞基因组中的特定靶序列的sgRNA、针对通用线性供体DNA上的通用靶序列的sgRNA，以引导Cas9切割细胞基因组中的特定靶序列和通用线性供体DNA上的通用靶序列。针对细胞基因组中的特定靶序列的sgRNA和针对通用线性供体DNA上的通用靶序列的sgRNA可以被包含在不同的载体中，也可以被包含在同一个载体中。
针对细胞基因组中的特定靶序列的sgRNA可以是一种sgRNA或更多种sgRNA，例如2种、 3种或更多种gRNA。这些多于一种的sgRNA可以分别针对细胞基因组中不同的特定靶序列，以实现同时对细胞基因组上的不同靶位点进行切割。当这些不同靶位点分别位于不同的基因上时，可以实现多个基因的敲除，例如2个、3个或更多个基因的敲除。具体而言，当进行多基因敲除时，可以向细胞中引入分别针对细胞基因组中的多个特定靶序列的多个sgRNA和针对通用线性供体DNA上的通用靶序列的sgRNA，以引导Cas9切割细胞基因组中的多个特定靶序列和通用线性供体DNA上的通用靶序列。所述多个特定靶序列分别位于不同的基因上，由此实现多基因敲除。所述分别针对细胞基因组中的多个特定靶序列的多个sgRNA可以被包含在不同的载体中，也可以被包含在同一个载体中。所述分别针对细胞基因组中的多个特定靶序列的多个sgRNA中的任何一个或更多个sgRNA与针对通用线性供体DNA上的通用靶序列的sgRNA可以被包含在不同的载体中，也可以被包含在同一个载体中。
根据本发明，所述通用线性供体DNA上的通用靶序列优选为5'-GTACGGGGCGATCATCCACA-3'或5'-AATCGACTCGAACTTCGTGT-3'。
优选地，本发明中，在使用Cas9/CRISPR系统产生基因敲除的情况下，可以将Cas9、sgRNA和线性供体DNA同时引入细胞，或者，例如，可以先将Cas9引入细胞，再将sgRNA和线性供体DNA引入细胞。在一些实施方案中，用包含Cas9的载体、包含sgRNA的载体和线性供体DNA共转染细胞。在另一些实施方案中，将Cas9和sgRNA在体外组装成蛋白和RNA复合体，并与线性供体DNA共转染细胞。在另一些实施方案中，将Cas9和sgRNA通过慢病毒稳定表达进细胞，并用线性供体DNA转染细胞。在其它的实施方案中，先将Cas9在细胞中稳定表达，再用包含sgRNA的载体和线性供体DNA共转染细胞。
在本发明提供的提供用于基因敲除的系统或试剂盒中，序列特异性核酸酶可以是蛋白质的形式或者编码核酸序列(例如mRNA或cDNA)的形式，例如是包含编码序列特异性核酸酶的核酸的质粒或病毒载体的形式。在使用Cas9/CRISPR系统的情况下，sgRNA可以是分离的RNA的形式，或者是包含编码sgRNA的序列和启动子的载体的形式，例如病毒载体或质粒载体。
本文所述的细胞可以是任何真核细胞，例如分离的动物细胞，例如全能细胞、多能细胞、成体干细胞、受精卵或体细胞等。在一些实施方案中，所述细胞是脊椎动物细胞。在一些实施方案中，所述细胞是哺乳动物细胞。在一些实施方案中，所述细胞是人细胞。在一些实施方案中，所述细胞是牛、山羊、绵羊、猫、狗、马、啮齿类动物、鱼、灵长类动物的细胞。在一些实施方案中，啮齿类动物包括小鼠、大鼠、兔。
本发明的方法可用于在细胞中的单个基因或多个基因上进行靶向基因敲除，例如靶向两个、三个、四个、五个或更多个基因敲除。针对多个基因的靶向基因敲除可以同时进行或先后进行。例如，可以将针对两个或更多个靶基因的序列特异性核酸酶或序列特异性核酸酶系统全部引入细胞后，再进行富集筛选。或者可以先将针对一个或更多个靶基因的序列特异性核酸酶或序列特异性核酸酶系统引入细胞后并进行富集筛选后，再将针对其它靶基因的序列特异性核酸酶或序列特异性核酸酶系统引入细胞后并进行富集筛选。针对不同的靶基因可以使用不同的标记物/标记物基因。例如，在使用Cas9/CRISPR系统产生基因敲除的情况下，可以向细胞中引入两个或更多个分别针对不同靶位点的sgRNA时，同时引入包含被这些sgRNA识别的靶序列的线性供体DNA，如前所述。当这些不同靶位点位于不同的基因上时，即可实现多个基因的基因敲除。还可以通过使用细胞基因组中两个或多个基因的共有序列设计sgRNA和线性供体DNA中的靶序列，此时可以向细胞中引入识别细胞基因组中单个特定靶位点的sgRNA，同时引入包含被所述sgRNA识别的靶序列的线性供体DNA，其中被所述sgRNA识别的靶序列是细胞基因组中两个或多个基因的共有序列，条件是所述共有序列与所述两个或多个基因的任何一个上与共有序列对应位置处的序列具有不超过一个碱基的差异。两个碱基的差异可能会破坏sgRNA的识别，如实施例7所证明的。
本发明的线性供体DNA进行的基因编辑所针对的靶基因没有特别限制，只要其能通过Cas9/CRISPR系统产生双链断裂。靶基因可以是外显子、内含子或调节序列，或其任意组合。
本发明中所使用的术语“包括”或“包含”表示“包括但不限于”、“基本上由……组成”或“由……组成”。
结合以下实施例和附图对本发明进行进一步说明，它们仅用于举例说明，并非要限制本发明的范围。如果没有特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular cloning:a laboratory manual,2001)，或按制造商提供的说明进行。
实施例1 利用线性供体DNA富集HeLa细胞中ANTXR1基因上的敲除事件
1.sgRNA的设计
设计两个靶向HeLa细胞中ANTXR1基因的第一外显子的sgRNA，通过T7E1测定验证它们在靶位点上产生缺失或插入突变(Indels)的效率，验证结果如表1所示。其中sgRNA1ANTXR1针对的靶序列在本实施例中被称为sg1，sgRNA2ANTXR1针对的靶序列在本实施例中被称为sg2。
表1 靶向HeLa细胞中ANTXR1基因的第一外显子的sgRNA
Figure PCTCN2017096510-appb-000001
2.线性供体DNA的构建
共构建了两种线性供体DNA(DonorANTXR1-sg2和DonorANTXR1-pg)，其结构参考图1a。
DonorANTXR1-sg2从5'至3'端分别包括：20bp的保护序列，sg2，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
DonorANTXR1-pg从5'至3'端分别包括：20bp的保护序列，sg1，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，sg2，20bp的保护序列。
作为对照的线性供体DNA(Donorno cut)从5'至3'端分别包括：20bp的保护序列，20bp的随机序列，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。其中随机序列不同于sg1或sg2。
3.转染
用表达Cas9的质粒、sgRNA2ANTXR1或pgRNAANTXR1、以及它们相应的供体共转染HeLa细胞，作为对照，单独用线性供体DNA(DonorANTXR1-sg2、DonorANTXR1-pg和Donorno cut)转染HeLa细胞，并加入嘌呤霉素进行抗性筛选，获得混合群落(pooled population)和单克隆(single clones)，用MTT(3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑)染色，结果如图1b所示。
从接受sgRNA2ANTXR1和它的相应供体DonorANTXR1-sg2，以及从接受pgRNAANTXR1和它的相应供体DonorANTXR1-pg的样品获得了许多具有嘌呤霉素抗性(puro+)的细胞克隆。仅用供体转染均只产生很少的嘌呤霉素抗性克隆，这可能是由于线性供体向染色体上的整合是稀少的且随机的。此外，对照供体Donorno cut与表达Cas9的质粒和sgRNA2ANTXR1共转染也未能产生显著量的puro+克隆(参见图1b最右图)，这表明sgRNA介导的Cas9在供体上的切割对于有效的供体整合是重要的。pgRNAANTXR1介导的双重切割DonorANTXR1-pg的整合比sgRNA2ANTXR1加上DonorANTXR1-sg2具有更高的效率；然而，无论用哪种线性供体DNA，都产生了足够的puro+克隆，可用于后续的突变体鉴别。
4.基因敲除效率的验证
另外用表达Cas9的质粒和sgRNA2ANTXR1或pgRNAANTXR1，加或不加它们相应的供体，共 转染HeLa细胞，利用嘌呤霉素(1μg/ml)筛选获得混合群落和单克隆，其中不加相应供体时与表达嘌呤霉素抗性基因的质粒共转染。由于HeLa细胞中ANTXR1基因的敲除导致细胞对嵌合炭疽毒素(PA/LFnDTA)的抗性[17]，用PA/LFnDTA(PA：150ng/ml；LFnDTA：100ng/ml)处理嘌呤霉素筛选获得的混合群落和单克隆，以比较线性供体DNA对ANTXR1敲除效率的影响。不同细胞用PA/LFnDTA处理后的图像如图2a所示。通过计算puro+混合群落中具有该毒素抗性的细胞的百分比确定ANTXR1敲除效率，如图1c所示，与单独使用sgRNA2ANTXR1或pgRNAANTXR1单独相比，线性供体DNA的使用将基因敲除效率提高了6-8倍。对puro+单克隆的线性供体整合的ANTRX1位点进行PCR验证，PCR扩增中使用的L1/R1引物序列如表2所示。结果如图2b所示，可以看出，从puro+细胞混合物中分离的大部分克隆在sgRNA靶向位点含有供体插入物(还参见图1d)。由图1d还可以看出，接近90％的携带供体片段的细胞是真正的基因敲除克隆。
表2 用于扩增HeLa细胞中线性供体整合的ANTRX1位点的引物
引物对 序列
L1/R1 5'-AAGCGGAGGACAGGATTGGG-3'/5'-CCTCTGTGGCCCTGGAGATG-3'
实施例2 利用线性供体DNA富集HeLa细胞中HBEGF基因上的敲除事件
由于具有单-或双-切割位点的供体都能够极大地提高对在靶位点具有修饰的细胞的选择，为方便起见，在本实施例中，仅使用单切割供体。
1.sgRNA的设计
设计两个靶向HeLa细胞中HBEGF基因的sgRNA，通过T7E1测定验证它们在靶位点上产生Indels的效率，验证结果如表3所示。其中sgRNA1HBEGF针对的靶序列在本实施例中被称为sg1，sgRNA2HBEGF针对的靶序列在本实施例中被称为sg2。
表3 靶向HeLa细胞中HBEGF基因的sgRNA
Figure PCTCN2017096510-appb-000002
2.线性供体DNA的构建
构建一种线性供体DNA(DonorHBEGF-sg1)，其结构参考图3a。
DonorHBEGF-sg1从5'至3'端分别包括：20bp的保护序列，sg1，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
3.转染
用表达Cas9的质粒、sgRNA1HBEGF、以及它相应的供体DonorHBEGF-sg1共转染HeLa细胞，作为对照，单独用供体DonorHBEGF-sg1转染HeLa细胞，并加入嘌呤霉素进行抗性筛选，获得混合群落(pooled population)和单克隆(single clones)，用MTT染色，结果如图3b所示。
与实施例1的结果类似，只有供体加上sgRNA获得大量的puro+克隆。这个结果再次证明供体插入取决于特异性的sgRNA/Cas9介导的DSBs。
4.基因敲除效率的验证
另外用表达Cas9的质粒和sgRNA1HBEGF，加或不加它相应的供体DonorHBEGF-sg1，共转染HeLa细胞，利用嘌呤霉素(1μg/ml)筛选获得混合群落和单克隆，其中不加相应供体时与表达嘌呤霉素抗性基因的质粒共转染。由于HBEGF基因编码白喉毒素(DT)受体，在HeLa细胞中将其敲除会导致细胞对DT的抗性[17]，用DT(40ng/ml)处理嘌呤霉素筛选获得的混合群落和单克隆，以比较线性供体DNA对HBEGF敲除效率的影响。不同细胞用DT处理后的图像如图3c所示。通过计算puro+混合群落中具有DT抗性的细胞的百分比确定HBEGF敲除效率，如图3d所示。由图3c和图3d可知，与单独使用sgRNA1HBEGF相比，线性供体DNA的使用大大提高了HBEGF基因敲除效率。
实施例3 利用线性供体DNA富集HEK293T细胞中HBEGF基因上的敲除事件
1.sgRNA的设计和线性供体DNA的构建
设计靶向HEK293T细胞中HBEGF基因的sgRNA2HBEGF，并构建线性供体DNA(DonorHBEGF-sg2)，供体从5'至3'端分别包括：20bp的保护序列，sg2，反向终止密码子，CMV启动子驱动的EGFP基因，正向终止密码子，20bp的保护序列，参见图4a。
2.基因敲除效率的验证
用表达Cas9的质粒和sgRNA2HBEGF，加或不加它相应的供体DonorHBEGF-sg2，共转染HEK293T细胞，通过FACS筛选细胞，加供体的组通过FACS筛选EGFP阳性细胞，不加供体的组通过FACS筛选mCherry阳性的细胞。用DT(40ng/ml)处理FACS选择的细胞，以比较线性供体DNA对HBEGF敲除效率的影响。不同细胞用DT处理后的图像如图4b所示。通过计算 EGFP阳性细胞中具有DT抗性的细胞的百分比确定HBEGF敲除效率，如图4d所示。与单独使用sgRNA2HBEGF相比，线性供体DNA的使用大大提高了HBEGF基因敲除效率。
实施例4 利用线性供体DNA富集HeLaOC细胞中ANTXR1基因上的敲除事件
1.HeLaOC细胞系的建立
根据已有的方法建立HeLaOC细胞系[17]，该细胞系稳定表达Cas9。
2.sgRNA的设计和线性供体DNA的构建
设计两个靶向HeLaOC细胞中ANTXR1基因的sgRNA(sgRNA1ANTXR1和sgRNA2ANTXR1)并构建三个线性供体DNA(DonorANTXR1-sg1、DonorANTXR1-sg2和DonorANTXR1-pg)，见图5a。其中sgRNA1ANTXR1针对的靶序列在本实施例中被称为sg1，sgRNA2ANTXR1针对的靶序列在本实施例中被称为sg2。
DonorANTXR1-sg1从5'至3'端分别包括：20bp的保护序列，sg1，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
DonorANTXR1-sg2从5'至3'端分别包括：20bp的保护序列，sg2，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
DonorANTXR1-pg从5'至3'端分别包括：20bp的保护序列，sg1，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，sg2，20bp的保护序列。
3.转染
用表达Cas9的质粒、sgRNA1ANTXR1或sgRNA2ANTXR1或pgRNAANTXR1、以及它们相应的供体共转染HeLaOC细胞，作为对照，单独用线性供体DNA(DonorANTXR1-sg1、DonorANTXR1-sg2和DonorANTXR1-pg)转染HeLaOC细胞，并加入嘌呤霉素进行抗性筛选，用MTT染色，结果如图5b所示。
与在HeLa细胞中的结果类似，只有供体加上sgRNA获得大量的puro+克隆。
4.基因敲除效率的验证
另外用表达Cas9的质粒和sgRNA1ANTXR1或sgRNA2ANTXR1或pgRNAANTXR1，加或不加它们相应的供体，共转染HeLaOC细胞，用嘌呤霉素(1μg/ml)进行筛选。用PA/LFnDTA处理筛选获得的细胞，不同细胞用PA/LFnDTA处理后的图像如图5c所示。通过计算puro+混合群落中具有该毒素抗性的细胞的百分比确定ANTXR1敲除效率，如图5d所示，与单独使用sgRNA相比，线性供体DNA的使用大大提高了ANTXR1基因敲除效率。
对puro+的单个克隆，对供体DonorANTXR1-sg1在ANTXR1基因上的整合位点进行PCR验证，发现puro+的大部分克隆在sgRNA靶向位点含有供体插入物(图5e和图5f)，大部分携带供体片段的细胞是真正的基因敲除克隆(图5f)。
对～500bp(长度相应于野生型ANTXR1基因)的PCR片段和～1.8kb(长度相应于野生型ANTXR1基因加上供体插入物)的PCR片段进行基因组测序，结果如表4所示。
表4 ～500bp的PCR片段和～1.8kb的PCR片段的基因组测序结果
靶位点：ANTXR1(Chr2,HeLaoc)
Figure PCTCN2017096510-appb-000003
从PCR验证结果(图5e)和测序结果(表4)可以看到，大部分克隆只含有一个供体插入。但是，在供体阳性克隆中，绝大多数等位基因在靶位点被编辑(或者说发生突变)，但是单独的sgRNA在不使用供体富集的情况下产生插入或缺失突变(indels)的效率很低。这一发现显然证明供体插入与sgRNA或pgRNA的作用密切相关。
5.供体对CRISPR/Cas系统的脱靶效应的影响
为了检查外部供体的使用是否会影响CRISPR/Cas系统的脱靶效果，通过splinkerette PCR分析找到全基因组的整合位点[35-37]。
本实施例中通过splinkerette PCR分析在嘌呤霉素选择之后验证单个克隆中和混合细胞克隆中脱靶的插入。如果在ANTXR1基因上具有正确的供体插入，用引物Splink2/R1和Splink2/R2扩增分别会产生711-和927-bp的产物(参见图6a)。
为了进行splinkerette PCR分析，我们随机选择了在HeLaOC细胞中靶向ANTXR1的10个具有供体插入的单个克隆和4个puro+混合克隆。根据splinkerette PCR结果(图6b)，与那些没有用供体转染的克隆类似，通过供体富集的单个克隆或混合群落没有可检测到的脱靶效应。
实施例5 利用线性供体DNA富集HeLaOC细胞中HBEGF基因上的敲除事件
1.sgRNA的设计和线性供体DNA的构建
设计两个靶向HeLaOC细胞中HBEGF基因的sgRNA(sgRNA1HBEGF和sgRNA2HBEGF)并构建三个线性供体DNA(DonorHBEGF-sg1、DonorHBEGF-sg2和DonorHBEGF-pg)，见图7a。其中sgRNA1HBEGF针对的靶序列在本实施例中被称为sg1，sgRNA2HBEGF针对的靶序列在本实施例中被称为sg2。
DonorHBEGF-sg1从5'至3'端分别包括：20bp的保护序列，sg1，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
DonorHBEGF-sg2从5'至3'端分别包括：20bp的保护序列，sg2，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
DonorHBEGF-pg从5'至3'端分别包括：20bp的保护序列，sg1，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，sg2，20bp的保护序列。
3.转染
用表达Cas9的质粒、sgRNA1HBEGF或sgRNA2HBEGF或pgRNAHBEGF、以及它们相应的供体共转染HeLaOC细胞，作为对照，单独用线性供体DNA(DonorHBEGF-sg1、DonorHBEGF-sg2和DonorHBEGF-pg)转染HeLaOC细胞，并加入嘌呤霉素进行抗性筛选，结果如图7b所示。
与在HeLa细胞中的结果类似，只有供体加上sgRNA获得大量的puro+克隆。
4.基因敲除效率的验证
对puro+的单个克隆，对供体DonorHBEGF-sg1在HBEGF基因上的整合位点进行PCR验证，PCR扩增中使用的L2/R2引物序列如表5所示。发现从puro+的大部分克隆在sgRNA靶向位点含有供体插入物(图7c和图7d)，大部分携带供体片段的细胞是真正的基因敲除克隆(图7d)。
表5 用于扩增HeLaOC细胞中线性供体整合的HBEGF位点的引物
引物对 序列
L2/R2 5'-GCCGCTTCGAAAGTGACTGG-3'/5'-GATCCCCCAGTGCCCATCAG-3'
实施例6 利用线性供体DNA在HeLaOC细胞中进行双基因敲除
1.sgRNA的设计
选定HeLaOC细胞中的两个靶基因：PSEN1和PSEN2，设计分别靶向这两个靶基因的两个sgRNA，通过T7E1测定验证它们在靶位点上产生Indels的效率，结果如表6所示。其中 sgRNAPSEN1针对的靶序列在本实施例中被称为sgPSEN1，sgRNAPSEN2针对的靶序列在本实施例中被称为sgPSEN2。
表6 靶向HeLaOC细胞中的PSEN1和PSEN2基因的sgRNA
Figure PCTCN2017096510-appb-000004
2.线性供体DNA的构建
构建两种类型的供体，一种具有两个单独的供体(DonorPSEN1+DonorPSEN2)，每个供体具有相应sgRNA的靶序列，另一种供体(DonorPSEN)在两端分别具有两个sgRNA靶序列，如图8a所示。DonorPSEN1或DonorPSEN2在其5'末端具有sgRNAPSEN1或sgRNAPSEN2切割位点。DonorPSEN1+PSEN2在5'端具有sgRNAPSEN1切割位点，在3'端具有sgRNAPSEN2切割位点。其中：
DonorPSEN1从5'至3'端分别包括：20bp的保护序列，sgPSEN1，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
DonorPSEN2从5'至3'端分别包括：20bp的保护序列，sgPSEN2，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
DonorPSEN从5'至3'端分别包括：20bp的保护序列，sgPSEN1，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，sgPSEN2，20bp的保护序列。
3、转染和基因敲除效率的验证
用表达Cas9的质粒、sgRNAPSEN1或sgRNAPSEN2共转染HeLaOC细胞，或者用表达Cas9的质粒、pgRNAPSEN共转染HeLaOC细胞，以在PSEN1和PSEN2基因的特定位点上产生indels。对indels产生效率进行T7E1分析[26](使用的引物见表7)，结果如图9a所示，它们的共转染都只显示出普通活性。
用表达Cas9的质粒、pgRNAPSEN、和DonorPSEN共转染HeLaOC细胞，或者用表达Cas9的质粒、pgRNAPSEN、和DonorPSEN1+DonorPSEN2共转染HeLaOC细胞，并加入嘌呤霉素进行抗性筛选，可以获得puro+克隆(见图9b)。与前面实施例的结果类似，供体加上pgRNA获得大量的puro+克隆。
对于每种转染结果，取puro+的单个克隆，对供体DonorPSEN和DonorPSEN1+DonorPSEN2在PSEN1和PSEN2基因上的整合位点进行PCR验证，PCR扩增中所使用的引物如表7所示，其中 L3/R3用于扩增PSEN1上的整合位点，L4/R4用于扩增PSEN2上的整合位点，PCR验证结果如图9c所示，两个基因上都含有供体插入的克隆用方框标出，选择克隆1和克隆2进一步进行基因组测序分析，两个克隆都显示出PSEN1和PSEN2的破坏(图8b和图8c)。
表7 用于扩增HeLaOC细胞中线性供体整合的PSEN1和PSEN2位点的引物
引物对 序列
L3/R3 5'-TGGTGTCTCAGGCGGTTCTA-3'/5'-TGAACTATGAGGCGCTGCAC-3'
L4/R4 5'-TGACTTTCGTGGCTATGCGT-3'/5'-CTAGCACCCAGGCATCCAAA-3'
实施例7 利用线性供体DNA在HeLaOC细胞中进行多基因敲除
1.靶基因的选择和sgRNA的设计
选择HeLaOC细胞中的HSPA基因家族，该基因家族有五个基因，HSAPA1A、HSPA1B、HSBA1L、HSPA6和HSPA2，它们具有同源性。设计同时靶向HSAPA1A、HSPA1B和HSBA1L的sgRNAHSPA，该sgRNA的靶序列与HSPA6上的相应序列有一个错配，与HSPA2有两个错配。如图8d所示。
2.线性供体DNA的构建
构建线性供体DonorHSPA，从5'至3'端分别包括：20bp的保护序列，sgHSPA，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列(图8d)。
3.转染和基因敲除效率的验证
用表达Cas9的质粒和sgRNAHSPA，加或不加其相应的供体DonorHSPA，共转染HeLaOC细胞，引发indels，并通过嘌呤霉素进行抗性筛选，。其中不加供体的组与表达嘌呤霉素抗性基因的质粒共转染。通过T7E1测定(使用的引物见表8)评价所有五个基因的indels效率，结果如图8e所示，与单独的sgRNAHSPA相比较，使用供体HSPA使得在HSPA1A位点的突变率提高大约5.5倍，在HSPA1B位点的突变率提高大约6.1倍，在HSPA1L位点的突变率提高大约3.4倍，在HSPA6位点的突变率提高大约6.6倍，有趣的是，在HSPA2基因上，无论是否使用供体，都没有检测到indels，表明两个错配完全破坏了sgRNAHSPA的识别，而且，更重要的是，使用供体进行选择没有增加脱靶效应的风险。
对与供体共转染和不与供体共转染获得的混合细胞中HSPA家族基因中的靶区域进行测序，结果如图10所示，结果表明无论在HSPA家族基因位点上是否有供体转染，细胞混合测序 结果都与T7E1测定的结果一致。
值得注意的是，T7E1测定证明所选择的群体高度富集携带有靶突变的细胞，与传统的不使用供体的方法相比，富集系数是大约753(5.5*6.1*3.4*6.6)。考虑到这种计算未考虑具有供体插入的基因，实际的效率提高甚至更高。
对具有嘌呤霉素抗性的单个克隆，在五个靶位点上进行PCR验证，用于扩增所有五个基因的靶位点使用的特异性引物(L5/R5，L6/R6，L7/R7，L8/R8，L9/R9)列在表8中。结果如图11a和11b所示。选择其中在至少两个靶基因上具有供体插入的六个克隆(在图中用方框标识出并分别编号为1-6)进行基因组序列分析，结果如图11b所示，克隆3在四个基因的相应位点上具有修饰：在HSPA1A、HSPA1B和HSPA1L上具有移码突变，产生完全的敲除，在HSPA6上具有两个框内突变(图8f)。
表8 用于扩增HeLaOC细胞中HSPA家族的五个基因靶位点的引物
引物对 序列
L5/R5 5'-GAGAGTGACTCCCGTTGTCC-3'/5'-ACATTGCAAACACAGGAAATTGAG-3'
L6/R6 5'-GTGTTGAGTTTCCGGCGTTC-3'/5'-TCGCTTGTTCTGGCTGATGT-3'
L7/R7 5'-GCACTCTCCCAAAACAGTATCTTA-3'/5'-GTGCCTCCACCCAGATCAAA-3'
L8/R8 5'-GGGTGAGGCGCAAAAGGATA-3'/5'-ACACCAGCGTCAATGGAGAG-3'
实施例8利用含有通用sgRNA的线性供体DNA富集SC-8细胞中CSPG4基因上的敲除事件
1、通用sgRNA的筛选
选择下述10个sgRNA作为筛选的候选序列，如下表所示：
sgRNA 靶序列(PAM)(5'至3') 预测的基因敲除效率
sgRNAUniversal_1 GTACGGGGCGATCATCCACA(CGG) 0.982784325
sgRNAUniversal_2 GCAAAAGTGGCATAAAACCG(CGG) 0.971302462
sgRNAUniversal_3 TATCGCTTCCGATTAGTCCG(CGG) 0.968382667
sgRNAUniversal_4 CTATCTCGAGTGGTAATGCG(CGG) 0.966411034
sgRNAUniversal_5 GTAGCTGCTGTAAATCGCAT(CGG) 0.963330804
sgRNAUniversal_6 TATACCAGACCACAGCGCCG(CGG) 0.962367571
sgRNAUniversal_7 GCACGAGGTGAACAGCCGCT(CGG) 0.960224565
sgRNAUniversal_8 ATGATATCTGACATGCAGCG(CGG) 0.95578653
sgRNAUniversal_9 AATCGACTCGAACTTCGTGT(CGG) 0.950640031
sgRNAUniversal_10 CGAATCGGAACTTTGTACCG(CGG) 0.948431616
2.线性供体DNA的构建
分别基于上述10种通用sgRNA构建10种线性供体DNA(DonorsgRNA_Universal_1～10-puro)。
这些线性供体DNA从5'至3'端分别包括：20bp的保护序列，sgRNAUniversal_1～10的靶序列，反向终止密码子，CMV启动子驱动的嘌呤霉素抗性基因，正向终止密码子，20bp的保护序列。
3.串联sgRNA质粒的构建
分别基于上述10种线性供体构建10种串联sgRNA质粒(PlasmidpgRNA_Universal_1～10)，其结构参见图12。
串联的两个sgRNA分别为sgRNACSPG4和sgRNAUniversal_1～10。其中sgRNACSPG4靶向TcdB毒素的受体CSPG4，sgRNAUniversal_1～10靶向对应的供体DNA(DonorsgRNA_Universal_1～10-puro)中的靶序列。
4、转染
转染实验所用细胞系为稳定表达Cas9的细胞系(SC-8)，用十种串联质粒PlasmidpgRNA_Universal_1～10和对应的供体DNA(DonorsgRNA_Universal_1～10-puro)共转染SC-8细胞，作为对照，单独用十种线性供体DNA(DonorsgRNA_Universal_1～10-puro)转染SC-8细胞。并加入嘌呤霉素进行抗性筛选，获得混合群落(pooled population)，筛选结果如下表所示。
Figure PCTCN2017096510-appb-000005
Figure PCTCN2017096510-appb-000006
根据上述结果，sgRNAUniversal_1、sgRNAUniversal_3、sgRNAUniversal_6和sgRNAUniversal_9四种sgRNA效果较好，因此后续实验采用这四种sgRNA对应的混合菌落作为实验对象。
4.基因敲除效率的验证
在四种混合菌落中加入TcdB毒素进行筛选，在23小时后观察细胞存活情况，实验结果如图13所示。可以看出，加入TcdB毒素23小时后，加入sgRNAUniversal_1和sgRNAUniversal_9实验组对应的细胞存活率明显高于其余两组，说明sgRNAUniversal_1和sgRNAUniversal_9的基因敲除效率更高。
以上实施例1-7中所使用的材料和方法如下：
细胞培养和转染
HeLa、HeLaOC和HEK293T细胞被保持在添加有10％胎牛血清(FBS，兰州百灵生物技术有限公司，兰州，中国)的Dulbecco’s modified Eagle’s培养基(DMEM,10-013-CV,Corning,Tewksbury,MA,USA)中，温度为37℃，通入5％CO2。为了进行转染，将所有细胞接种于6孔板上，并用X-tremeGENE HP(06366546001,Roche,Mannheim,Germany)转染，根据供应商的说明进行。简单地说，将2μg DNA和4μl X-tremeGENE HP加入到200μl Opti-MEM I Reduced Serum Medium(31985088,Thermo Fisher Scientific,Grand Island,NY,USA)中。在室温将混合物温育15分钟，然后加入到细胞中。
表达gRNA的质粒的克隆
对于表达sgRNA的质粒，单独设计每个sgRNA编码序列的寡核苷酸(见表9)，并进行合成(北京睿博兴科生物技术有限公司)。
表9 用于sgRNA或pgRNA构建的引物
Figure PCTCN2017096510-appb-000007
用1×TE将寡核苷酸溶解至浓度为10μM，用TransTaq HiFi BufferⅡ(K10222，北京全式金生物技术有限公司)混合成对寡核苷酸，加热至95℃保持3分钟，然后缓慢冷却至4℃。在37℃将这些退火的寡核苷酸对磷酸化30分钟，加热失活后，使用“Golden Gate”法将产物连接到sgRNA骨架载体中。对于表达pgRNA的质粒，用含有两个gRNA编码序列的引物扩增gRNA的scaffold序列和U6启动子(表5)，然后纯化PCR产物并使用“Golden Gate”法连接到sgRNA骨架载体中。与先前报道的sgRNA骨架载体[17]相比，本发明的sgRNA骨架载体对sgRNA骨架进行了修改[38]，并且用mCherry编码序列取代了EGFP序列。
T7E1测定
使用DNeasy Blood&Tissue kit(69504,Qiagen,Hilden,Germany)提取基因组DNA，对含有gRNA靶序列的基因组区域进行PCR扩增。测定中使用的引物序列如表2、表5、表7、表8所示。 所使用的引物序列如在50μl体系中取300-500ng的PCR产物与10×NEB Buffer2混合，95℃加热3分钟后缓慢冷却到室温。所得产物加入0.5μl T7E1在37℃温育15min，进行琼脂糖凝胶电泳，电泳图用Image J图像分析软件分析条带切割的效率，指示sgRNA产生Indels的效率。
线性供体构建
预先生成含有CMV驱动的嘌呤霉素抗性基因或EGFP基因的供体和终止密码子的序列，并克隆到pEASY-T5-Zero克隆载体(CT501-02，北京全式金生物技术有限公司)中，作为通用模板。使用含有sgRNA切割靶位点和保护序列的引物扩增模板，引物序列如表10所示。
表10 用于线性供体构建的引物
Figure PCTCN2017096510-appb-000008
Figure PCTCN2017096510-appb-000009
基于NHEJ的供体插入和细胞选择
在HeLaOC细胞中，用1μg纯化的线性供体PCR产物和1μg sgRNA/pgRNA转染细胞，转染后两周，用1μg/ml的嘌呤霉素处理细胞。在HeLa和HEK293T细胞中，用1μg供体和0.5μg sgRNA/pgRNA以及0.5μg Cas9质粒转染细胞。然后在转染后两周用1μg/ml的嘌呤霉素处理细胞，或者通过荧光激活的细胞分选(FACS)确定EGFP阳性，这取决于使用哪种类型的供体。
Splinkerette PCR
Splinkerette PCR方法先前已有报道(Potter,C.J.&Luo,L.Splinkerette PCR for mapping transposable elements in Drosophila.PLoS One 5,e10168(2010)；Uren,A.G.et al.A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.Nat Protoc 4,789-798(2009)；Yin,B.&Largaespada,D.A.PCR-based procedures to isolate insertion sites of DNA elements.Biotechniques 43,79-84(2007))。所使用的引物和接头(adaptor)序列如表11所示。
表11 用于Splinkerette PCR的引物
Figure PCTCN2017096510-appb-000010
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Claims (10)

  1. 通用供体构建体，所述通用供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的通用靶序列，所述通用靶序列含有可被Cas9核酸酶切割的靶位点；位于两端的保护序列；
    其中所述表达盒包含由启动子驱动的标记物基因；
    其中所述通用靶序列在待进行基因敲除的细胞的基因组中不存在。
  2. 权利要求1的通用供体构建体，其是线性供体DNA，优选是双链线性供体DNA。
    优选地，所述线性供体DNA仅在5'端或3'端具有所述通用靶序列，或者所述线性供体DNA在两端分别具有所述通用靶序列。
    更优选地，其中所述标记物基因是抗生素抗性基因或荧光蛋白基因。
  3. 权利要求1-2任一项的通用供体构建体，其中所述保护序列的长度为5-30bp，最优选20bp。
    优选地，所述通用靶序列含有5'-GTACGGGGCGATCATCCACA-3'或5'-AATCGACTCGAACTTCGTGT-3'。
  4. 在细胞中产生基因敲除的方法，所述方法包括以下步骤：
    (1)向细胞中引入：
    (a)Cas9核酸酶；
    (b)识别细胞基因组中特定靶序列的gRNA；
    (c)通用供体构建体，其中所述通用供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的通用靶序列，所述通用靶序列含有可被Cas9核酸酶切割的靶位点；位于两端的保护序列；
    其中所述表达盒包含由启动子驱动的标记物基因；
    其中所述通用靶序列在待进行基因敲除的细胞的基因组中不存在；
    (d)识别线性供体DNA中包含的通用靶序列的gRNA；
    (2)所述线性供体DNA通过非同源末端连接被插入到细胞基因组中的特定靶位点；
    (3)筛选所述标记物表达阳性的细胞。
  5. 权利要求4的方法，其中所述通用供体构建体是线性供体DNA，优选是双链线性供体DNA。
    更优选地，所述线性供体DNA仅在5'端或3'端具有所述通用靶序列，或者所述线性供体DNA在两端分别具有所述通用靶序列。
  6. 权利要求4-5任一项的方法，其中所述识别细胞基因组中特定靶序列的gRNA是一种gRNA，或者是多于一种的识别细胞基因组中不同靶序列的gRNA。
    优选地，所述识别细胞基因组中特定靶序列的gRNA是sgRNA，和/或所述识别线性供体DNA中包含的通用靶序列的gRNA是sgRNA。
    更优选地，其中所述识别细胞基因组中特定靶序列的sgRNA和所述识别线性供体DNA中包含的通用靶序列的sgRNA位于相同的载体中；或者
    所述识别细胞基因组中特定靶序列的sgRNA和所述识别线性供体DNA中包含的通用靶序列的sgRNA位于不同的载体中。
  7. 权利要求4-6任一项的方法，其中所述标记物基因是抗生素抗性基因或荧光蛋白基因。
    优选地，其中通过药物抗性筛选细胞，或者通过FACS方法筛选细胞。
    优选地，其中所述保护序列的长度为5-30bp，最优选20bp。
    更优选地，其中所述通用靶序列含有5'-GTACGGGGCGATCATCCACA-3'或5'-AATCGACTCGAACTTCGTGT-3'。
  8. 用于基因敲除的系统或试剂盒，其中包括：
    (1)Cas9核酸酶或能够表达Cas9核酸酶的载体或细胞；
    (2)识别细胞基因组中特定靶序列的gRNA；
    (3)通用供体构建体，其中所述通用供体构建体是线性供体DNA或可在细胞中被切割产生线性供体DNA，所述线性供体DNA由中间向两端依次包含：表达盒；位于表达盒5'端的由反向终止密码子组成的短的序列延伸和位于表达盒3'端的由正向终止密码子组成的短的序列延伸；位于5'端和/或3'端的通用靶序列，所述通用靶序列含有可被Cas9核酸酶切割的靶位点；位于两端的保护序列；
    其中所述表达盒包含由启动子驱动的标记物基因；
    其中所述通用靶序列在待进行基因敲除的细胞的基因组中不存在；
    (4)识别线性供体DNA中包含的通用靶序列的gRNA。
  9. 权利要求8的系统或试剂盒，其中所述通用供体构建体是线性供体DNA，优选是双链线性供体DNA。
    优选地，其中所述线性供体DNA仅在5'端或3'端具有所述通用靶序列，或者所述线性供体DNA在两端分别具有所述通用靶序列。
    更优选地，其中所述识别细胞基因组中特定靶序列的gRNA是一种gRNA，或者是多于一种的识别细胞基因组中不同靶序列的gRNA。
    还更优选地，其中所述识别细胞基因组中特定靶序列的gRNA是sgRNA，和/或所述识别线性供体DNA中包含的通用靶序列的gRNA是sgRNA。
    特别优选地，其中所述识别细胞基因组中特定靶序列的sgRNA和所述识别线性供体DNA中包含的通用靶序列的sgRNA位于相同的载体中；或者
    所述识别细胞基因组中特定靶序列的sgRNA和所述识别线性供体DNA中包含的通用靶序列的sgRNA位于不同的载体中。
  10. 权利要求9的系统或试剂盒，其中所述标记物基因是抗生素抗性基因或荧光蛋白基因。
    优选地，其中所述保护序列的长度为5-30bp，最优选20bp。
    还优选地，其中所述通用靶序列含有5'-GTACGGGGCGATCATCCACA-3'或5'-AATCGACTCGAACTTCGTGT-3'。
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