CN109385421A

CN109385421A - Gene knockout method

Info

Publication number: CN109385421A
Application number: CN201710672040.4A
Authority: CN
Inventors: 魏文胜; 陈欧; 陈一欧; 周悦欣; 张鸿敏; 袁鹏飞; 刘源
Original assignee: Because Ji Boya (beijing) Biotechnology Co Ltd; Peking University
Current assignee: Because Ji Boya (beijing) Biotechnology Co Ltd; Peking University
Priority date: 2017-08-08
Filing date: 2017-08-08
Publication date: 2019-02-26

Abstract

The present invention relates to universal donor construct and gene knockout method and its system and kits.The marker gene for including in universal donor construct is inserted on the double-strand break position in cellular genome by gene knockout method of the invention by non-homologous end joining, it is enriched with the cell that gene is knocked by the marker of expression, improves the efficiency for generating gene knockout by sequence specific nuclease.Universal donor construct of the invention includes General Linear donor dna, general target sequence on the General Linear donor dna comprising that can be cut by sequence specific nuclease, the general target sequence is not present in the genome of the cell of pending gene knockout, therefore, when carrying out gene knockout, without specially constructing matching used linear donor dna, directly using the gRNA of General Linear donor dna and targeting the General Linear donor dna.

Description

Gene knockout method

Technical field

The present invention relates to gene editing technologies, and in particular to gene knockout method.

Background technique

Gene editing technology revolutionizes the experimental study to gene function.Three kinds of main technologies, ZFN (zinc finger core Sour enzyme) [1], TALENs (transcriptional activator sample effector nuclease) [2-4] and CRISPR/Cas9 system [5-7], using not Same mechanism generates sequence-specific duplex fracture (double-strand breaks, DSBs) and then causes natural reparation System completes sequence specific sex modification [8,9].These technologies study the dynamic and reality of [10], chromosomal foci in functional gene When imaging [11,12], the correction [13] of disease mutation, gene therapy [14] etc. have a wide range of applications.CRISPR/ Cas9 system becomes especially welcome because of its high efficiency and easily operated.CRISPR/Cas9 system is originally by bacterial immune system System is used to resist exogenous virus or plasmid, in II class CRISPR system, guidance incision of the Cas9 endonuclease in sgRNA Cut double-stranded DNA, cause genome double-strand break, using cellular genome reparation unstability generate repair mistake (base Missing or insertion), to generate the effect of gene editing.

Although CRISPR/Cas9 system in based on design and sequence-specific genome research have it is preceding it is all not Some advantages, but the gene editing that CRISPR/Cas9 system causes still falls within rare event in cell colony, to obtain Real gene monoclonal to be edited needs to carry out cumbersome labour, therefore the system technically still has challenge Property, for simple task as generation gene knockout in mammalian cells [15].It has been carried out various Make great efforts improve generate gene knockout scheme efficiency, such as integrate CRISPR/Cas9 system with permanently express Cas9 and SgRNA [16], in advance generate stablize expression Cas9 cell line [17], enhancing non-homologous end joining (NHEJ) approach [18], By destroying the independent gene for being able to achieve specific drug selection simultaneously to be enriched with gene-targeting event [19] and using generation Reason reports sub (surrogate reporter) enrichment gene knockout [20,21].But various disadvantages limit these technologies Extensive use.In particular, generating polygenes knockout in mammalian cells is still being difficult to complete of the task.Work as use When conventional method, even knock out individual gene is also prolonged, heavy and high risk task [22] sometimes, because They lack effective enrichment to the rare clone modified containing target gene.

If the destruction of target gene can lead to the character mutation that can be used in enrichment, gene knockout can be readily available Clone, such as Hela CSPG4^-/-Cell imparts the resistance to clostridium difficile (Clostridium difficile) toxin B [23].But the strategy can not be general.Conventional method is to want the plasmid of cotransfection expression antibiotic resistance or fluorescin [23,24], but this method will not be enriched with a small amount of cell containing targeting modification.

It has been reported that external source dsDNA segment may be integrated into the dyeing position with DSBs by different repair mechanisms Point on.Need to construct long flanking sequence by homologous recombination (HR) reparation, integration efficiency is lower, and connects by nonhomologous end The integration efficiency [27,28] for connecing (Non-Homologous end joing, NHEJ) DNA reparation is repaired usually above homologous recombination Multiple [29].Existing research mediates the insertion of external source linear donor dna using the NHEJ that CRISPR/Cas causes, to reach gene The purpose [30-34] knocked in.

Summary of the invention

The present invention provides donor construct and gene knockout method and its system and kit.Clpp gene of the invention Except method is enriched with using the marker gene for including in the donor construct by the cell of gene knockout, raising passes through sequence The efficiency of specific nucleic acid enzyme generation gene knockout.

According to an aspect of the present invention, donor construct is provided, the donor construct is linear donor dna or can be Linear donor dna is generated by cutting in cell, the linear donor dna successively includes from centre to both ends: expression cassette；It is located at The end expression cassette 5' is extended by the molecular short sequence of reversed termination codon and positioned at the close by forward direction termination of the end expression cassette 3' The molecular short sequence of code extends；Target sequence positioned at the end 5' and/or the end 3', the target sequence contains can be by the sequence The target site that specific nucleic acid digestion is cut；Protection sequence positioned at both ends；Wherein the expression cassette includes to be driven by promoter Marker gene.

In the present invention, the linear donor dna is double stranded donor dna.

In preferred embodiments, the donor construct is linear donor dna.

In some embodiments, the sequence specific nuclease is Zinc finger nuclease (ZFN).

In other embodiments, the sequence specific nuclease is transcriptional activator sample effector nuclease (TALEN)。

In other embodiments, the sequence specific nuclease is Cas9 nuclease.

In other embodiments, the sequence specific nuclease is NgAgo nuclease.

In some embodiments, the linear donor dna only has target sequence at the end 5' or the end 3'.

In some embodiments, the linear donor dna is respectively provided with target sequence at both ends.

In some embodiments, the target sequence at both ends is identical in the linear donor dna.

In some embodiments, the target sequence at both ends is different in the linear donor dna.In further embodiment In, the different target sequences at both ends are from identical gene in the linear donor dna.In other further embodiments In, the different target sequences at both ends are from different genes in the linear donor dna.

In preferred embodiments, the marker gene is antibiotics resistance gene or fluorescence protein gene.

In preferred embodiments, the length for protecting sequence is 5-30bp, most preferably 20bp.

According to another aspect of the present invention, provide in cell generate gene knockout method, the method includes with Lower step:

(1) sequence specific nuclease and donor structure in particular target site in energy incising cell genome are introduced into cell Build body；

Wherein the donor construct is linear donor dna or can generate linear donor dna by cutting in cell, described Linear donor dna successively includes from centre to both ends: expression cassette；Positioned at being made of reversed terminator codon for the end expression cassette 5' Short sequence extend and being extended by the molecular short sequence of positive termination codon positioned at the end expression cassette 3'；Positioned at the end 5' And/or the target sequence at the end 3', the target sequence contain the target site that can be cut by the sequence specific nuclease；Positioned at two The protection sequence at end；The expression cassette includes the marker gene driven by promoter；

Wherein linear donor dna is inserted into the particular target site in cellular genome by non-homologous end joining；

(2) cell of the marker representation positive is screened.

In the present invention, the linear donor dna is double stranded donor dna.

In preferred embodiments, the donor construct is linear donor dna.

In other embodiments, the sequence specific nuclease is Cas9 nuclease.

In preferred embodiments, the method also includes specific target site in identification cellular genome is introduced into cell The guide RNA (gRNA) of point, the target sequence in linear donor dna include the target site identified by the gRNA.

In some embodiments, the gRNA is sgRNA.

In a more preferred embodiment, single special in identification cellular genome the method also includes being introduced into cell The sgRNA in targeting site, the target sequence comprising the target site identified by the sgRNA be located at linear donor dna the end 5' and/ Or the end 3'.In some embodiments, the target sequence comprising the target site identified by the sgRNA is from cellular genome In individual gene.In some embodiments, the target sequence of the target site comprising being identified by the sgRNA is cytogene The consensus sequence of two or more genes in group, condition are any the one of the consensus sequence and described two or multiple genes A upper and consensus sequence corresponding position sequence has the difference no more than a base.

In other further preferred embodiments, the method also includes being introduced into identification cellular genome into cell Two sgRNA in two particular target sites on one gene separately include two target position identified by described two sgRNA Two target sequences of point are located in two linear donor dnas, or are located at the both ends of the same linear donor dna.

In other further preferred embodiments, the method also includes being introduced into identification cellular genome into cell Two or more sgRNA in two or more particular target sites are separately included by described two or multiple sgRNA identification Two or more target sequences of two or more target sites are located at the both ends or position of the same linear donor dna In different linear donor dnas.Wherein two or more particular target sites are located at difference in the cellular genome Gene on.

In other embodiments, the sequence specific nuclease is NgAgo nuclease.

In preferred embodiments, specific in identification cellular genome the method also includes being introduced into cell The guide DNA (gDNA) of target site, wherein the target sequence in linear donor dna includes the target site identified by the gDNA.

In the present invention, the gene knockout can be individual gene knockout or polygenes knocks out.The polygenes knocks out The knockout of two genes or more gene, such as the knockout of three, four, five or more genes.

In a preferred embodiment, cell is screened by drug resistance.

In another preferred embodiment, cell is screened by FACS method.

According to another aspect of the present invention, the system or kit for gene knockout are provided, including: it can cut The sequence specific nuclease and donor construct in particular target site in cellular genome；

Wherein the donor construct is linear donor dna or can generate linear donor dna by cutting in cell, described Linear donor dna successively includes from centre to both ends: expression cassette；Positioned at being made of reversed terminator codon for the end expression cassette 5' Short sequence extend and being extended by the molecular short sequence of positive termination codon positioned at the end expression cassette 3'；Positioned at the end 5' And/or the target sequence at the end 3', the target sequence contain the target site that can be cut by the sequence specific nuclease；Positioned at two The protection sequence at end；The expression cassette includes the marker gene driven by promoter.

In the present invention, linear donor dna is double stranded donor dna.

In some embodiments, the donor construct is linear donor dna.In other embodiments, described Donor construct is annular donor construct and can generate linear donor dna by cutting in cell.

In other embodiments, the sequence specific nuclease is Cas9 nuclease.

In preferred embodiments, the system or kit further include particular target site in identification cellular genome SgRNA, wherein the target sequence in linear donor dna includes the target site identified by the sgRNA.

In some embodiments, the gRNA is sgRNA.

In other embodiments, the sequence specific nuclease is NgAgo nuclease.

In preferred embodiments, the system or kit further include particular target site in identification cellular genome GDNA, wherein the target sequence in linear donor dna includes the target site identified by the gDNA.

In the present invention, the cutting is to generate double-strand break (DSBs).

According to another aspect of the present invention, universal donor construct is provided, the universal donor construct is linearly to supply Body DNA can generate linear donor dna by cutting in cell, and the linear donor dna successively includes from centre to both ends: Expression cassette；Positioned at being extended and by the molecular short sequence of reversed termination codon positioned at the end expression cassette 3' for the end expression cassette 5' Extended by the molecular short sequence of positive termination codon；General target sequence positioned at the end 5' and/or the end 3', the general target Sequence contains the target site that can be cut by Cas9 nuclease；Protection sequence positioned at both ends；

Wherein the expression cassette includes the marker gene driven by promoter；

Wherein it is not present in the genome of cell of the general target sequence in pending gene knockout.

In some embodiments, the universal donor construct is linear donor dna.

In some embodiments, the linear donor dna is double stranded donor dna.

In some embodiments, the linear donor dna only has the general target sequence at the end 5' or the end 3'.

In some embodiments, the linear donor dna is respectively provided with the general target sequence at both ends.

In preferred embodiments, the general target sequence in the universal donor construct contains

5'-GTACGGGGCGATCATCCACA-3' or 5'-AATCGACTCGAACTTCGTGT-3'.

(1) it is introduced into cell:

(a) Cas9 nuclease；

(b) gRNA of specific target sequence in cellular genome is identified；

(c) universal donor construct, wherein the universal donor construct is linear donor dna or can be cut in cell Generation linear donor dna is cut, the linear donor dna successively includes from centre to both ends: expression cassette；Positioned at the end expression cassette 5' Extended and by the molecular short sequence of reversed termination codon positioned at the molecular by positive termination codon of the end expression cassette 3' Short sequence extends；General target sequence positioned at the end 5' and/or the end 3', the general target sequence contains can be by Cas9 nuclease The target site of cutting；Protection sequence positioned at both ends；

Wherein the expression cassette includes the marker gene driven by promoter；

Wherein it is not present in the genome of cell of the general target sequence in pending gene knockout；

(d) gRNA for the general target sequence for including in linear donor dna is identified.

(2) linear donor dna is inserted into the particular target site in cellular genome by non-homologous end joining；

(3) cell of the marker representation positive is screened.

In some embodiments, the donor construct is linear donor dna.

In some embodiments, the linear donor dna is double stranded donor dna.

In some embodiments, the linear donor dna all has the general target sequence at both ends.

In some embodiments, the gRNA of specific target sequence can be a kind of gRNA in the identification cellular genome, Or a variety of gRNA for identifying different target sequences in cellular genomes, such as in 2 kinds, 3 kinds or more identification cellular genomes The gRNA of different target sequences.The difference target sequence can be located in the same gene, or can be located at different genes In.When the different target sequences are located in different genes, the knockout of multiple genes may be implemented.

Therefore, in the present invention, the gene knockout can be individual gene knockout or polygenes knocks out.The polygenes strikes Except the knockout for being two genes or more gene, such as the knockout of three, four, five or more genes.

In some embodiments, the gRNA of specific target sequence is sgRNA in the identification cellular genome.

In some embodiments, the gRNA for the general target sequence for including in the identification linear donor dna is sgRNA.

In some embodiments, the sgRNA of specific target sequence and the identification are linear in the identification cellular genome The sgRNA for the general target sequence for including in donor dna is located in identical carrier.

In some embodiments, the sgRNA of specific target sequence and the identification are linear in the identification cellular genome The sgRNA for the general target sequence for including in donor dna is located in different carriers.

In a preferred embodiment, cell is screened by drug resistance.

In another preferred embodiment, cell is screened by FACS method.

5'-GTACGGGGCGATCATCCACA-3' or 5'-AATCGACTCGAACTTCGTGT-3'.

According to another aspect of the present invention, the system or kit for gene knockout are provided, including:

(1) Cas9 nuclease or the carrier or cell of Cas9 nuclease can be expressed；

(2) gRNA of specific target sequence in cellular genome is identified；

(3) universal donor construct, wherein the universal donor construct is linear donor dna or can be cut in cell Generation linear donor dna is cut, the linear donor dna successively includes from centre to both ends: expression cassette；Positioned at the end expression cassette 5' Extended and by the molecular short sequence of reversed termination codon positioned at the molecular by positive termination codon of the end expression cassette 3' Short sequence extends；General target sequence positioned at the end 5' and/or the end 3', the general target sequence contains can be by Cas9 nuclease The target site of cutting；Protection sequence positioned at both ends；

Wherein the expression cassette includes the marker gene driven by promoter；

(4) gRNA for the general target sequence for including in linear donor dna is identified.

In some embodiments, linear donor dna is double stranded donor dna.

In some embodiments, the donor construct is linear donor dna.

In other embodiments, the donor construct is annular donor construct and can be produced in cell by cutting Raw linear donor dna.

In some embodiments, the gRNA of specific target sequence and the linear confession of the identification in the identification cellular genome The gRNA for the general target sequence for including in body DNA is located in identical carrier.

In some embodiments, the gRNA of specific target sequence and the linear confession of the identification in the identification cellular genome The gRNA for the general target sequence for including in body DNA is located in different carriers.

In preferred embodiments, the target sequence in the universal donor construct contains

5'-GTACGGGGCGATCATCCACA-3' or 5'-AATCGACTCGAACTTCGTGT-3'.

The present invention can be effective by marker by being inserted into marker gene on the cutting target site of gene knockout Being enriched with has the rare clone for generating gene knockout.Targeting of the present invention for the gene of sgRNAs difficult design, and needing It is particularly useful in the case where targeting several gene knockouts simultaneously.This method facilitates various generation DNA double chain fractures Gene editing system, especially CRISPR system is in gene and its broader applications of the field of biomedicine of function.

Detailed description of the invention

Fig. 1 is to contain Cas9/gRNA target on the ANTXR1 gene being enriched in HeLa cell by puromycin selection Donor design and experimental verification to the cell of mutation.(a) sgRNA- or pgRNA target of the linear donor on ANTXR1 gene To the schematic diagram knocked in based on NHEJ in site, cleavage site sg1 on genomic locus and linear donor with gRNA and Sg2, terminator codon are marked with * * *, and arrow is directed toward the direction of reading frame.(b) it is transfected with donor (add gRNA or gRNA is not added) Cell puromycin-resistant clone MTT dyeing.(c) (added (dark bars) with sgRNA/pgRNA or be not added (light item) Their corresponding donors) the ANTXR1 knockout rate of HeLa cell of transfection compares, and ANTXR1 knockout rate is expressed as with PA/ The percentage of the cell of LFnDTA resistance.It is thin with puromycin (1 μ g/ml) selection before carrying out PA/LFnDTA Analysis of Resistance Born of the same parents.Error line indicates s.d. (n=3), t-test, P < 0.001 * * P < 0.01, * * *.(d) different gRNAs and its confession are used Body is enriched with the summary that ANTXR1 knocks out cell.

Fig. 2 is that the puromycin-resistant mediated by donor selects to be enriched with ANTXR1 knockout in mictium and monoclonal The experimental verification of cell.(a) image that difference HeLa groups of cells is handled with or without the use of PA/LFnDTA.With sgRNA or PgRNA adds or is not added their corresponding linear donor transfections to obtain cell mixing, and scale bar is 200 μm.(b) to purine The PCR verifying in the site ANTXR1 of the linear donor integration of chloramphenicol resistance (puro+) monoclonal, is cloned from sgRNA2_ANTXR1/ Donor_ANTXR1-sg2(left side) or pgRNA_ANTXR1/Donor_ANTXR1-pgThe HeLa cell of (right side) transfection obtains.

Fig. 3 is the donor design and experimental verification that HEBGF destructive insident in enrichment HeLa cell is selected by puromycin. (a) the donor design of HBEGF gene is targeted.(b) linear donor Donor is used_HBEGF-sg1(adding or be not added Cas9/sgRNA) transfection Cell in puromycin-resistant clone MTT dyeing.(c) difference HeLa groups of cells with or without the use of DT (40ng/ml) at The image of reason.With sgRNA (sgRNA1_HBEGF) (add or be not added its corresponding linear donor (Donor_HBEGF-sg1)) transfect and mixed Cell is closed, scale bar is 200 μm.(d) sgRNA (sgRNA1 is used_HBEGF), expression Cas9 plasmid and contain puromycin-resistant The reporter plasmid of gene (light item), or plasmid and linear donor (Donor with sgRNA, expression Cas9_HBEGF-sg1) (deep Vitta) transfection HeLa cell HBEGF knockout rate, HBEGF knockout rate is expressed as the percentage to the cell with DT resistance Than.Cell is selected with puromycin (1 μ g/ml) before analyzing DT resistance.Error line indicates s.d. (n=3), t- P < 0.001 test, * * *.

Fig. 4 is the donor design and experimental verification that HBEGF destructive insident in HEK293T cell is enriched with by EGFP.(a) target It is designed to the donor of HBEGF gene.(b) figure that difference HEK293T groups of cells is handled with or without the use of DT (40ng/ml) Picture.With sgRNA (sgRNA2_HBEGF) (add or be not added their corresponding linear donor (Donor_HBEGF-sg2)) transfection acquisition mixing is carefully Born of the same parents, 200 μm of scale bar.(c) with the sgRNA (sgRNA2 of expression mCherry_HBEGF) plasmid and expression Cas9 plasmid (light color Item), or with sgRNA, express Cas9 plasmid and linear donor (Donor_HBEGF-sg2, EGFP) (dark bars) transfection The HBEGF knockout rate of HEK293T cell, HBEGF knockout rate are indicated with the percentage of the cell resistant to DT.It is analyzing Cell is selected with FACS before DT resistance.Error line indicates s.d. (n=3), t-test, P < 0.05 * * *.

Fig. 5 is that enrichment HeLa is selected by puromycin_OCThe donor of ANTXR1 destructive insident in cell designs and experiment is tested Card.(a) the donor design of ANTXR1 is targeted.Donor contains a sgRNA cleavage site (Donor at the end 5'_ANTXR1-sg1Or Donor_ANTXR1-sg2) or contain there are two gRNAs (Donor at both ends_ANTXR1-pg).It (b) is (to be added gRNA with donor or be not added GRNA) MTT of puromycin-resistant clone is dyed in the cell transfected.(c) difference HeLa_OCGroups of cells is with or without the use of PA/ The image of LFnDTA processing.The corresponding linear donor transfection for being added with sgRNA or pgRNA or it being not added obtains cell mixing, ratio Ruler is 200 μm.(d) add or be not added the HeLa that their corresponding donor transfects with sgRNAs_OCThe ANTXR1 knockout rate of cell, ANTXR1 knockout rate is expressed as the percentage of the cell with PA/LFnDTA resistance.Before analyzing PA/LFnDTA resistance Cell is selected with puromycin (1 μ g/ml).Error line indicates s.d. (n=3), t-test, P < 0.001 * * *.(e) to purine The PCR verifying in the site ANTXR1 of the linear donor integration of chloramphenicol resistance monoclonal.(f) using different gRNAs and they Donor is enriched with the summary that ANTXR1 knocks out cell.

Fig. 6 is in HeLa_OCDonor insertion miss the target commenting by splinkerette PCR (spPCR) analysis in cell Estimate.(a) design for connector (adaptor) and primer of spPCR analysis.Splink1 and Splink2 primer and connector sequence Column matching, primer R1 and R2 and linear donor sequences match.(b) spPCR reaction result.

Fig. 7 is that enrichment HeLa is selected by puromycin_OCThe donor of HBEGF destructive insident in cell designs and experiment is tested Card.(a) the donor design of HBEGF is targeted.Donor contains a sgRNA cleavage site (Donor at the end 5'_HBEGF-sg1Or Donor_HBEGF-sg2) or contain there are two gRNAs (Donor at both ends_HBEGF-pg).(b) with donor (adding or be not added sgRNA/pgRNA) The MTT dyeing that puromycin-resistant is cloned in the cell of transfection.(c) linear donor of puromycin-resistant monoclonal is integrated The site HBEGF PCR verifying.(d) the total of cell enrichment is knocked out using the HBEGF of different gRNAs and their donor Knot.

Fig. 8 is in HeLa_OCA step generates the donor design and experimental verification of two or more gene knockouts in cell.(a) The schematic diagram knocked in based on NHEJ of sgRNA- or pgRNA target site of the linear donor on PSEN1 and PSEN2 gene. (b-c) in genome PSEN1 and PSEN2 partial coding sequence, contain sgRNA coding region (underlining) and mutation etc. The sequencing analysis of position gene.1 (b) is cloned from pgRNA_PSEN1+PSEN2/Donor_PSEN1+PSEN2The HeLa of transfection_OCCell. 2 (c) are cloned from pgRNA_PSEN1+PSEN2/Donor_PSEN1+Donor_PSEN2The HeLa of transfection_OCCell.The core of shadow region Thuja acid represents the PAM sequence that guidance Cas9 carries out DNA identification and cutting.Dotted line indicates missing, and high letter indicates that nucleotide is inserted Enter, the light grey arrows in background indicate the direction of CMV promoter in donor.(d) Multiple Sequence Alignment of HSPA gene family point Analysis, shows its consensus sequence, targets the sgRNA of the consensus sequence of five HSPA family genes, and contain more bases for being enriched with Because of the General Linear donor (Donor of the cell of mutation_HSPA) design.Black shade nucleotide represents institute, and there are five HSPA genes Consensus sequence.Dark grey shade nucleotide represents the consensus sequence of three or four HSPA genes, light grey shade nucleotide Represent non-shared nucleotide.(e) lacking and there are Donor_HSPAUnder conditions of, after having carried out puromycin selection, sgRNA_HSPAThe indels caused on five target genes.Error line indicates s.d. (n=3), t-test, * * P < 0.01, * * * P < 0.001.(f) HSPA1A, HSPA1B in the genome of the HeLa clone 3 containing sgRNA target area (underlining), The partial coding sequence of HSPA1L and HSPA6 gene.Clone 3 is from sgRNA_HSPA/Donor_HSPAThe HeLa of transfection_OCCarefully Born of the same parents.The nucleotide of dash area represents PAM sequence, and dotted line represents missing.Light grey arrows in background indicate in donor CMV promoter direction.

Fig. 9 is HeLa_OCPSEN1 and PSEN2sgRNA efficiency rating and single clonal operator in cell.(a) pass through T7E1 points Analysis in the site PSEN1 and PSEN2 to sgRNA_PSEN1、sgRNA_PSEN2And pgRNA_PSENCaused indels efficiency is evaluated. Error line indicates s.d. (n=3).(b) (add or be not added pgRNA with donor_PSEN) transfection cell in puromycin-resistant clone MTT dyeing.(c) PSEN1 (L3/R3) and PSEN2 (L4/R4) two of the linear donor integration of puromycin-resistant monoclonal The PCR result in a site.

Figure 10 is the sequencer map of the target region of HSPA family gene in the cell mixing with or without donor transfection.sgRNA Target site is marked with shade, does not include the target region containing donor insertion in these sequencing analysis.

Figure 11 is five HSPA family genes, on the target site of HSPA1A, HSPA1B, HSPA1L, HSPA6 and HSPA2 The monoclonal of donor insertion identifies.(a) puromycin-resistant monoclonal there are five the linear donor integration on gene loci PCR verification result.(b) donor is inserted into the total of result in four gene locis HSPA1A, HSPA1B, HSPA1L, HSPA6 Knot.

Figure 12 is the experiment flow figure that gene knockout is carried out using the donor comprising general sgRNA.

Figure 13 is the gene knockout efficiency verifying that gene knockout is carried out using the donor comprising general sgRNA.

Specific embodiment

The present invention provides new donor construct and gene knockout method, this method is improved logical using linear donor dna Cross the efficiency that sequence specific nuclease generates gene knockout.It can be by comprising at least one on linear donor dna of the invention The target site of sequence specific nuclease cutting.Include according in the target site design linear donor dna in cellular genome Target site, the sequence specific nuclease for making it possible to target site in incising cell genome can also be cut in linear donor dna The target site for including.After sequence specific nuclease and donor construct are introduced cell, sequence specific nuclease is in cell When middle particular target site generates double-strand break (DSBs), at least one target position for including in linear donor dna can be cut simultaneously Point, it is possible thereby to which linear donor dna is made to pass through non-homologous end joining (Non-Homologous end with higher efficiency Joining, NHEJ) approach is inserted on the target site cut in cellular genome, then by the marker to cell It is selected, can effectively be enriched with and generate the cell of gene knockout because the particular target site in genome is by cutting, greatly Improve the efficiency that gene knockout is generated by sequence specific nuclease in ground.

According to the target site for including in the target site design linear donor dna in cellular genome, linear confession obtained Body DNA is specific linear donor, when needing the different target sites in cellular genome to carry out gene knockout, needs root According to the matching used linear donor dna of the sequence construct of the target site.Therefore, in order to advanced optimize the present invention, inventor exists General Linear donor dna is further provided in the present invention, including on the General Linear donor dna can be by sequence-specific The general target sequence of nuclease cutting, the general target sequence are not present in the genome of the cell of pending gene knockout, i.e., In the genome of the cell of pending gene knockout there is no it is identical with the general target sequence can be by the sequence specific Property nuclease cutting sequence.In the case, after sequence specific nuclease and General Linear donor dna being introduced cell, Sequence specific nuclease includes in General Linear donor dna when particular target site generates double-strand break (DSBs) in cell General target sequence also by identify the target sequence general gRNA cut by the sequence specific nuclease, at this time linearly Donor dna still can pass through non-homologous end joining (Non-Homologous end joining, NHEJ) with higher efficiency Approach is inserted on the target site cut in cellular genome, is then selected by the marker cell, can be with It is effectively enriched with and generates the cell of gene knockout because the particular target site in genome is by cutting, equally can greatly mention The efficiency of high pass sequence specific nuclease generation gene knockout.Target sequence in the General Linear donor dna with wait strike Except gene is unrelated, it can be used as knockout of the general donor for the different target gene in different cells, can be improved The efficiency of gene knockout is generated by sequence specific nuclease.General Linear donor dna is particularly useful for using Cas9/ CRISPR system carries out the case where gene knockout, which targets target sequence using gRNA (preferably sgRNA), when progress base When because knocking out, the gRNA for particular target site in cellular genome need to be only constructed, it is matching used linear without specially constructing Donor dna is thus reduced directly using the gRNA of General Linear donor dna and targeting the General Linear donor dna Operation complexity improves efficiency.

It has been reported that if one in homoallele is modified, the frequency of mutation of target allele usually compared with High [25,26].Therefore, while not wishing to be limited by theory, but inventor speculates, if it is possible in target allele Specific site on one is inserted into donor, and the clone for the marker gene for selecting expression donor to contain, it may be possible to be enriched with that The rare event that a little all allele are all modified.

In the present invention, " gene knockout " is the forfeiture that gene function is realized by gene editing.The gene usually pursued Knocking out effect is two allele while knocking out, and corresponds to albumen loss of function at this time, obtains Knockout cells system.If Only one allele is knocked, then albumen can also play partial action, and only protein function is lowered.Utilize the present invention Linear donor dna and method of the invention, can effectively be enriched with the cell that two allele are all knocked.

Donor construct of the invention is double-stranded DNA.Donor construct of the invention itself can be linear donor DNA.Or donor construct of the invention can be the circular DNA molecule comprising linear donor dna, when being introduced in cell When middle, linear donor dna is generated by cutting in cell.Annular donor construct is cut in cell generates linear donor dna Method be well known in the art.For example, can be in the end the 5' upstream and the end 3' downstream of linear donor dna in annular construct Further include the cleavage site of another sequence specific nuclease.

In the method for the invention, it may further include to cell and introduce another sequence specific nuclease, the sequence Column specific nucleic acid enzyme cuts the end the 5' upstream of linear donor dna and the sequence in the end 3' downstream in annular construct in cell, Thus linear donor dna is generated.

In linear donor dna of the invention, " reversed terminator codon " refers to the direction of the codon and readding for expression cassette Frame it is contrary." positive terminator codon " refers to that the direction of the direction of the codon and the reading frame of expression cassette is identical. The effect of terminator codon is: no matter linear donor is by positive or be reversely inserted into genome, and two three terminator codons are all It can stop endogenous and exogenous gene expression.

" protection sequence " can be arbitrary sequence in linear donor dna of the invention, preferably protection sequence and same linear Target sequence in donor dna is different.The length of protection sequence can be 5-30bp, preferably 20bp.The effect of protection sequence is to protect The target sequence protected in linear donor dna is not cut by the enzyme (such as exonuclease) in cell.

" marker gene " described herein refers to that it expresses any marker gene that can be selected or be enriched with, i.e., When the marker gene is expressed in cell, it can select and be enriched with by certain way to express the thin of the marker gene Born of the same parents.Marker gene for use in the present invention includes but is not limited to the fluorescence protein gene that can be sorted with FACS after expression, Perhaps it can be identified and be passed through immune by corresponding antibody after can use the resistant gene or expression that antibiotic is screened Dyeing or magnetic bead adsorb the protein gene screened.Resistant gene for use in the present invention includes but is not limited to be directed to kill rice Pest rhzomorph (Blasticidin), Geneticin (Geneticin, G-418), hygromycin (Hygromycin B), mycophenolic acid (Mycophenolic Acid), puromycin (Puromycin), bleomycin (Zeocin) or neomycin (Neomycin) Resistant gene.Fluorescence protein gene for use in the present invention includes but is not limited to blue fluorescent protein (Cyan Fluorescent Protein), green fluorescent protein (Green Fluorescent Protein), yellow fluorescence protein (Yellow Fluorescent Protein), orange fluorescent protein (Orange Fluorescent Protein), red fluorescent protein (Red Fluorescent Protein), remote red fluorescent protein (Far-Red Fluorescent Protein) can be cut Change the gene of fluorescin (Switchable Fluorescent Proteins).

The example of sequence specific nuclease includes Zinc finger nuclease (ZFN).Zinc finger nuclease is non-naturally occurring, people The endonuclease of work transformation, is made of zinc finger protein structural domain and non-specific nucleic acid inscribe enzyme domains.Zinc finger protein knot Structure domain is composed in series by a series of Cys2-His2 zinc finger proteins, and each zinc finger protein identifies and combines 3 ' to 5 ' direction DNA chain Upper one special triplet base and a base in 5 ' to 3 ' directions.Multiple zinc finger proteins can be together in series to be formed One zinc finger protein group identifies one section of special base sequence, has very strong specificity.It is connected with zinc finger protein group non- Specific endonucleases shear domain from the DNA of 96 amino acid residue compositions of FokI c-terminus.Each FokI monomer and one A zinc finger protein group be connected constitute a ZFN, identify specific site, when two recognition sites at a distance of it is appropriate apart from when (6 ~8bp), two monomer ZFN interactions generate digestion function, to achieve the purpose that DNA fixed point shearing.For target sequence 8~10 Zinc finger domains are designed, these zinc structural domains are connected on DNA nuclease, can realize the double-strand break of target sequence (Double strand breaks, DSBs), and then DSBs repair mechanism is induced, the specific site in genome is determined To transformation.

Another example of sequence specific nuclease includes transcriptional activator sample effector nuclease (TALEN).Transcription swashs Being sample effector nuclease is mainly composed of the DNA binding structural domain of Fok I inscribe enzyme domains and TALE albumen. TALE albumen contains the repetition peptide fragment of multiple 33-35 amino acid compositions, and each peptide fragment can identify a base. As ZFN, TALEN can also be such that DNA target sequence is broken, and DSBs be formed, and then activate DNA damage repair mechanism, to genome Carry out fixed point transformation.

Another example of sequence specific nuclease system for use in the present invention includes Cas9/CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats, the regular intervals of cluster it is short Palindrome repetitive sequence) system.The DNA that Cas9/CRISPR system is instructed using RNA, which is combined, cuts target DNA progress sequence-specific It cuts, is tied by crRNA (CRISPR-derived RNA) by base pairing and tracrRNA (trans-activating RNA) Conjunction forms tracrRNA/crRNA compound, this compound guides nuclease Cas9 albumen on the target sequence matched with crRNA Specific location shear double-stranded DNA.Being typically situated in genome PAM with the target sequence of crRNA pairing, (former spacer region is neighbouring Motif) upstream site (NNG) about 20 nucleotide sequence.

Cas9 albumen needs by guide RNA the cutting of target site.Term " guide RNA " is also known as gRNA (guide RNA), gRNA generally includes nucleotide complementary with target sequence on crRNA and is formed by crRNA and tracrRNA base pairing RNA bracket (Scaffold), can identify with crRNA pairing target sequence.GRNA can form complex with Cas9 albumen And by Cas9 protein band to target sequence and cut target site therein.

GRNA is usually used in the form of sgRNA (single guide RNA).SgRNA is also known as " single-stranded guide RNA ", is A RNA chain made of crRNA and trancrRNA fusion.

Another example of sequence specific nuclease system for use in the present invention include NgAgo nuclease and its gDNA.NgAgo nuclease can be to combine with the single-stranded guide DNA (gDNA) of the end 5' phosphorylation, to the target complementary with the gDNA Sequence is cut, and DNA double chain is caused to be broken.

Linear donor dna of the invention can only have target sequence at one end, can also be respectively provided with target sequence at both ends. The target sequence at linear donor dna both ends can be different.When needs are cut on two different target sites in cellular genome And when generating gene knockout, two linear donor dnas can be provided, each linear donor dna separately includes a corresponding target Sequence, can also provide a linear donor dna, and every one end of the linear donor dna includes a corresponding target sequence.When When needing to be cut on multiple and different target sites in cellular genome and generate gene knockout, right quantity can be provided Linear donor dna, the one or both ends of each linear donor dna separately include one in multiple and different corresponding target sequences. Such as linear donor dna identical with target site quantity can be provided, each linear donor dna separately includes one accordingly Target sequence.Or the linear donor dna less than target site quantity can be provided, wherein the two of all or part of linear donor dna End separately includes one in multiple and different corresponding target sequences, and other linear donor dnas each separately include other respective target sequences One in column.

For containing the General Linear donor dna of general target sequence, then can have in its any one or both ends and contain The general target sequence, the target sequence of this General Linear donor dna is independent of target position to be cut off in cellular genome Point, thus can predominately be suitable for any one of cellular genome target site, any two target site or appoint The case where what more target site is cut and generates gene knockout,.

" general target sequence " of the present invention refers to the sequence that can be cut by sequence specific nuclease, but the general target Sequence is not present in the genome of the cell of pending gene knockout, that is to say, that in the cell of pending gene knockout The sequence that can be cut by the sequence specific nuclease identical with the general target sequence is not present in genome, it is general The target sequence that can be cut by same sequence specific nuclease present on target sequence and cellular genome is different.Contain this The linear donor dna of general target site specificity all no for the target site in cellular genome, therefore can generally fit For constructing spy without being directed to the target site of the gene for needing to knock out i.e. thereon in the gene knockout to gene any in cell Fixed linear donor dna.

Sequence specific nuclease can in the form of protein or with its nucleic acid sequence encoding (such as mRNA or CDNA form) is introduced into cell.The nucleic acid of coded sequence specific nucleic acid enzyme can be contained in plasmid or viral vectors It is introduced into cell, such as cell is introduced by transfection.The nucleic acid of coded sequence specific nucleic acid enzyme can also be worn by electricity The modes such as hole, liposome, microinjection are delivered directly in cell.

Donor construct can be suitable for the method being introduced into nucleic acid in cell delivering by any, such as by transfecting quilt It is introduced into cell.

In the case where generating gene knockout using Cas9/CRISPR system and NgAgo nuclease, also by sgRNA or GDNA introduces cell.Can deliver sgRNA or gDNA by any method RNA or DNA being introduced into cell that is suitable for. SgRNA can be introduced into cell in the form of isolated RNA.Any in-vitro transcription system as known in the art can be used By the way that the sgRNA come preparative separation is transcribed in vitro.It can also be incited somebody to action by the inclusion of the sequence of coding sgRNA and the carrier of promoter SgRNA introduces cell.The carrier can be viral vectors or plasmid.The mode for introducing cell can be transfection.

Two or more sgRNA for being directed to different target sites respectively can be introduced, into cell to guide Cas9 to cut It is cut on the different target sites of two or more in cellular genome and generates gene knockout.It is the two or more SgRNA can be contained in different carriers, also can be contained in the same carrier, such as include a pair of of gRNA The carrier of (paired gRNA), or the carrier comprising more sgRNA.

In the method for the invention, when introducing two or more sgRNA for being directed to different target sites respectively into cell When, while introducing comprising by the linear donor dna of these sgRNA target sequence identified.Since linear donor dna can only exist Target sequence is contained at the end 5' or the end 3', can also contain target sequence, the quantity of sgRNA and the number of linear donor dna respectively at both ends Amount can be different, can be the corresponding linear donor dna of a sgRNA, are also possible to corresponding two of two sgRNA linearly Donor dna.

When carrying out gene knockout using Cas9/CRISPR system and General Linear donor dna of the invention, in addition to that will lead to Cell is introduced with linear donor dna and Cas9 nuclease, to be also introduced into cell for the particular target sequence in cellular genome The sgRNA of column, for the sgRNA of the general target sequence on General Linear donor dna, to guide Cas9 incising cell genome In specific target sequence and General Linear donor dna on general target sequence.For the specific target sequence in cellular genome It sgRNA and can be contained in different carriers for the sgRNA of the general target sequence on General Linear donor dna, it can also To be comprised in the same carrier.

It can be a kind of sgRNA or more sgRNA, such as 2 for the sgRNA of the specific target sequence in cellular genome Kind, 3 kinds or more gRNA.These more than one sgRNA can be respectively for particular target sequence different in cellular genome Column simultaneously cut the different target sites in cellular genome with realizing.When these difference target sites are located at difference Gene on when, the knockout of multiple genes, such as the knockout of 2,3 or more genes may be implemented.Specifically, working as When carrying out polygenes knockout, it can be introduced into cell respectively for the multiple of multiple specific target sequences in cellular genome SgRNA and sgRNA for the general target sequence on General Linear donor dna, to guide in Cas9 incising cell genome General target sequence on multiple specific target sequences and General Linear donor dna.The multiple specific target sequence is located at difference Gene on, be achieved in polygenes knockout.It is described respectively for the multiple of multiple specific target sequences in cellular genome SgRNA can be contained in different carriers, also can be contained in the same carrier.It is described to be directed to cell base respectively Because any one of multiple sgRNA of multiple specific target sequences in group or more sgRNA be directed to General Linear donor The sgRNA of general target sequence on DNA can be contained in different carriers, also can be contained in the same carrier.

According to the present invention, the general target sequence on the General Linear donor dna is preferably

5'-GTACGGGGCGATCATCCACA-3' or 5'-AATCGACTCGAACTTCGTGT-3'.

Preferably, the present invention in, using Cas9/CRISPR system generate gene knockout in the case where, can by Cas9, SgRNA and linear donor dna introduce cell simultaneously, alternatively, for example, cell first can be introduced Cas9, then by sgRNA and line Property donor dna introduce cell.In some embodiments, with the carrier comprising Cas9, the carrier comprising sgRNA and linear confession Body DNA cotransfection cells.In other embodiments, Cas9 and sgRNA are assembled into albumen and RNA complex in vitro, And with linear donor dna cotransfection cells.In other embodiments, Cas9 and sgRNA is stablized by slow virus and is expressed Cell is transfected into cell, and with linear donor dna.In other embodiments, Cas9 is first stablized to expression in cell, Again with the carrier comprising sgRNA and linear donor dna cotransfection cells.

It is provided in system or kit for gene knockout provided by the invention, sequence specific nuclease can be The form of the form or nucleic acid sequence encoding (such as mRNA or cDNA) of protein, e.g. comprising coded sequence specificity The plasmid of the nucleic acid of nuclease or the form of viral vectors.Using Cas9/CRISPR system, sgRNA be can be The form of isolated RNA, or comprising encode sgRNA sequence and promoter carrier form, such as viral vectors or Plasmid vector.

Cell as described herein can be any eukaryocyte, such as isolated zooblast, such as totipotent cell, multipotency Cell, adult stem cell, fertilized eggs or body cell etc..In some embodiments, the cell is vertebrate cells.? In some embodiments, the cell is mammalian cell.In some embodiments, the cell is people's cell.One In a little embodiments, the cell is the cell of ox, goat, sheep, cat, dog, horse, rodent, fish, primate. In some embodiments, rodent includes mouse, rat, rabbit.

Method of the invention can be used on the individual gene in cell or multiple genes carrying out targeted gene disruption, such as Targeting two, three, four, five or more gene knockout.For multiple genes targeted gene disruption can simultaneously into Capable or successively progress.For example, the sequence specific nuclease or sequence-specific of two or more target genes can will be directed to After nucleic acid enzyme system is fully incorporated cell, then carry out enrichment isolation.Or it will can first be directed to one or more target genes Sequence specific nuclease or sequence specific nuclease system introduce after cell and after carrying out enrichment isolation, then will be directed to it The sequence specific nuclease or sequence specific nuclease system of its target gene introduce after cell and carry out enrichment isolation.Needle Different target genes can be used different marker/marker genes.For example, being generated using Cas9/CRISPR system In the case where gene knockout, when can introduce the sgRNA that two or more are directed to different target sites respectively into cell, together When introduce comprising by the linear donor dna of these sgRNA target sequence identified, as previously described.When these difference target sites are located at When on different genes, the gene knockout of multiple genes can be realized.It can also be by using two in cellular genome or more Target sequence in the consensus sequence design sgRNA and linear donor dna of a gene, it is thin can to introduce identification into cell at this time The sgRNA in single particular target site in born of the same parents' genome, while introducing comprising by the linear confession of the sgRNA target sequence identified Body DNA, wherein being the consensus sequence of two or more genes in cellular genome, condition by the sgRNA target sequence identified It is that any one upper sequences with consensus sequence corresponding position of the consensus sequence and described two or multiple genes has No more than the difference of a base.The difference of two bases may destroy the identification of sgRNA, as embodiment 7 is proved.

The targeted target gene of the gene editing that linear donor dna of the invention carries out is not particularly limited, as long as its energy Double-strand break is generated by Cas9/CRISPR system.Target gene can be exon, introne or adjusting sequence or it is any Combination.

Term " includes " used in the present invention or "comprising" indicate " including but not limited to ", " substantially by ... group At " or " by ... form ".

Invention is further explained with attached drawing with the following Examples, they are only used for for example, not really wanting It limits the scope of the invention.If do not specialized, embodiment is according to conventional laboratory conditions, such as Sambrook molecule Cloning: A Laboratory Manual (Sambrook J&Russell DW, Molecular cloning:a laboratory manual, 2001), or by what manufacturer provided illustrate to carry out.

Embodiment 1 utilizes the knockout event on ANTXR1 gene in linear donor dna enrichment HeLa cell

The design of 1.sgRNA

The sgRNA for designing the First Exon of ANTXR1 gene in two targeting HeLa cells, is measured by T7E1 and is verified They generate the efficiency of missing or insertion mutation (Indels) on target site, and verification result is as shown in table 1.Wherein sgRNA1_ANTXR1For target sequence in the present embodiment be referred to as sg1, sgRNA2_ANTXR1For target sequence in the present embodiment In be referred to as sg2.

Table 1 targets the sgRNA of the First Exon of ANTXR1 gene in HeLa cell

2. the building of linear donor dna

Two kinds of linear donor dna (Donor are constructed altogether_ANTXR1-sg2And Donor_ANTXR1-pg), structural reference Fig. 1 a.

Donor_ANTXR1-sg2The end from 5' to 3' respectively include: the protection sequence of 20bp, sg2, reversed terminator codon, CMV The puromycin resistance gene of promoter driving, positive terminator codon, the protection sequence of 20bp.

Donor_ANTXR1-pgThe end from 5' to 3' respectively include: the protection sequence of 20bp, sg1, reversed terminator codon, CMV are opened The puromycin resistance gene of mover driving, positive terminator codon, the protection sequence of sg2,20bp.

Linear donor dna (Donor as control_{no cut}) end from 5' to 3' respectively include: the protection sequence of 20bp, The random sequence of 20bp, reversed terminator codon, the puromycin resistance gene of CMV promoter driving, positive termination codon Son, the protection sequence of 20bp.Wherein random sequence is different from sg1 or sg2.

3. transfection

With plasmid, the sgRNA2 of expression Cas9_ANTXR1Or pgRNA_ANTXR1And their corresponding donor cotransfection HeLa are thin Born of the same parents individually use linear donor dna (Donor as control_ANTXR1-sg2、Donor_ANTXR1-pgAnd Donor_no _cut) transfection HeLa it is thin Born of the same parents, and puromycin is added and carries out resistance screening, obtain mictium (pooled population) and monoclonal (single Clones), dyed with MTT (3- (4,5- dimethylthiazole -2- base) -2,5- diphenyl bromination tetrazolium), as a result as shown in Figure 1 b.

From receiving sgRNA2_ANTXR1The corresponding donor Donor with it_ANTXR1-sg2, and from receiving pgRNA_ANTXR1With it Corresponding donor Donor_ANTXR1-pgSample obtain many cell clones with puromycin-resistant (puro+).Only with confession Body transfection only generates seldom puromycin-resistant clone, this may be that integration due to linear donor on chromosome is It is rare and random.In addition, control donor Donor_{no cut}With the plasmid and sgRNA2 of expression Cas9_ANTXR1Cotransfection is not yet The puro+ clone (referring to Fig. 1 b most right figure) of significant quantity can be generated, this shows cutting of the Cas9 of sgRNA mediation on donor The integration of effective donor is important.pgRNA_ANTXR1The dual cutting Donor mediated_ANTXR1-pgIntegration ratio sgRNA2_ANTXR1In addition Donor_ANTXR1-sg2It has higher efficiency；However, all being produced no matter with which kind of linear donor dna Enough puro+ clones, can be used for subsequent mutant and identify.

4. the verifying of gene knockout efficiency

In addition with the plasmid and sgRNA2 of expression Cas9_ANTXR1Or pgRNA_ANTXR1, add or be not added their corresponding donors, altogether Transfection HeLa cell is screened using puromycin (1 μ g/ml) and obtains mictium and monoclonal, wherein when corresponding donor is not added With the plasmid co-transfection of expression puromycin resistance gene.Since the knockout of ANTXR1 gene in HeLa cell leads to cell pair The resistance [17] of chimeric anthrax toxin (PA/LFnDTA), with PA/LFnDTA (PA:150ng/ml；LFnDTA:100ng/ml) The mictium and monoclonal that puromycin screening obtains are handled, the shadow of efficiency is knocked out to compare linear donor dna to ANTXR1 It rings.Treated that image is as shown in Figure 2 a with PA/LFnDTA for different cells.By calculating there is the poison in puro+ mictium The percentage of the cell of plain resistance determines that ANTXR1 knocks out efficiency, as illustrated in figure 1 c, with exclusive use sgRNA2_ANTXR1Or pgRNA_ANTXR1It individually compares, gene knockout efficiency is improved 6-8 times by the use of linear donor dna.To puro+ monoclonal The site ANTRX1 of linear donor integration carries out PCR verifying, and L1/R1 primer sequence used in PCR amplification is as shown in table 2.Knot Fruit is as shown in Figure 2 b, it can be seen that the major part separated from puro+ cell mixture is cloned in sgRNA target site and contains Donor insert (sees also Fig. 1 d).It can also be seen that the cell close to 90% carrying donor fragment is real by Fig. 1 d Gene knockout clone.

Table 2 is used to expand the primer in the site ANTRX1 that linear donor is integrated in HeLa cell

Primer pair	Sequence
		L1/R1	5'-AAGCGGAGGACAGGATTGGG-3'/5'-CCTCTGTGGCCCTGGAGATG-3'

Embodiment 2 utilizes the knockout event on HBEGF gene in linear donor dna enrichment HeLa cell

There is the thin of modification in target site since the donor with mono- or double- cleavage site can be greatlyd improve For convenience in the present embodiment, single cutting donor is used only in the selection of born of the same parents.

The design of 1.sgRNA

The sgRNA for designing HBEGF gene in two targeting HeLa cells verifies them on target site by T7E1 measurement The efficiency of Indels is generated, verification result is as shown in table 3.Wherein sgRNA1_HBEGFFor target sequence claimed in the present embodiment For sg1, sgRNA2_HBEGFFor target sequence in the present embodiment be referred to as sg2.

Table 3 targets the sgRNA of HBEGF gene in HeLa cell

2. the building of linear donor dna

Construct a kind of linear donor dna (Donor_HBEGF-sg1), structural reference Fig. 3 a.

Donor_HBEGF-sg1The end from 5' to 3' respectively include: the protection sequence of 20bp, sg1, reversed terminator codon, CMV are opened The puromycin resistance gene of mover driving, positive terminator codon, the protection sequence of 20bp.

3. transfection

With plasmid, the sgRNA1 of expression Cas9_HBEGFAnd its corresponding donor Donor_HBEGF-sg1Cotransfection HeLa cell, As control, donor Donor is individually used_HBEGF-sg1Transfection HeLa cell, and puromycin is added and carries out resistance screening, it is mixed It is gregarious to fall (pooled population) and monoclonal (single clones), it is dyed with MTT, as a result as shown in Figure 3b.

Similar with the result of embodiment 1, only donor obtains a large amount of puro+ clone plus sgRNA.This result is again Prove that donor insertion depends on the DSBs that the sgRNA/Cas9 of specificity is mediated.

4. the verifying of gene knockout efficiency

In addition with the plasmid and sgRNA1 of expression Cas9_HBEGF, add or be not added its corresponding donor Donor_HBEGF-sg1, corotation Contaminate HeLa cell, screened using puromycin (1 μ g/ml) and obtain mictium and monoclonal, wherein when corresponding donor is not added with Express the plasmid co-transfection of puromycin resistance gene.It is thin in HeLa due to HBEGF gene encoding diphtheria toxin (DT) receptor The resistance [17] that will lead to cell to DT is knocked out in born of the same parents, the mixing obtained with DT (40ng/ml) processing puromycin screening Group and monoclonal knock out the influence of efficiency to compare linear donor dna to HBEGF.Different cells DT treated image As shown in Figure 3c.Percentage by calculating the cell in puro+ mictium with DT resistance determines that HBEGF knocks out efficiency, As shown in Figure 3d.By Fig. 3 c and Fig. 3 d it is found that with sgRNA1 is used alone_HBEGFIt compares, the use of linear donor dna mentions significantly High HBEGF gene knockout efficiency.

Embodiment 3 utilizes the knockout event on HBEGF gene in linear donor dna enrichment HEK293T cell

The design of 1.sgRNA and the building of linear donor dna

The sgRNA2 of HBEGF gene in design targeting HEK293T cell_HBEGF, and construct linear donor dna (Donor_HBEGF-sg2), donor end from 5' to 3' respectively include: the protection sequence of 20bp, sg2, reversed terminator codon, CMV are opened The EGFP gene of mover driving, positive terminator codon, the protection sequence of 20bp, referring to fig. 4 a.

2. the verifying of gene knockout efficiency

With the plasmid and sgRNA2 of expression Cas9_HBEGF, add or be not added its corresponding donor Donor_HBEGF-sg2, cotransfection HEK293T cell screens cell by FACS, adds the group of donor to screen EGFP positive cell by FACS, the group of donor is not added The cell of the mCherry positive is screened by FACS.The cell selected with DT (40ng/ml) processing FACS, to compare linear donor DNA knocks out the influence of efficiency to HBEGF.Treated that image is as shown in Figure 4 b with DT for different cells.It is positive by calculating EGFP The percentage of cell in cell with DT resistance determines that HBEGF knocks out efficiency, as illustrated in fig. 4 c.With exclusive use sgRNA2_HBEGFIt compares, the use of linear donor dna substantially increases HBEGF gene knockout efficiency.

Embodiment 4 is enriched with HeLa using linear donor dna_OCKnockout event in cell on ANTXR1 gene

1.HeLa_OCThe foundation of cell line

HeLa is established according to existing method_OCCell line [17], the cell line stablize expression Cas9.

The design of 2.sgRNA and the building of linear donor dna

Design two targeting HeLa_OCSgRNA (the sgRNA1 of ANTXR1 gene in cell_ANTXR1And sgRNA2_ANTXR1) and structure Build three linear donor dna (Donor_ANTXR1-sg1、Donor_ANTXR1-sg2And Donor_ANTXR1-pg), see Fig. 5 a.Wherein sgRNA1_ANTXR1For target sequence in the present embodiment be referred to as sg1, sgRNA2_ANTXR1For target sequence in the present embodiment In be referred to as sg2.

Donor_ANTXR1-sg1The end from 5' to 3' respectively include: the protection sequence of 20bp, sg1, reversed terminator codon, CMV The puromycin resistance gene of promoter driving, positive terminator codon, the protection sequence of 20bp.

3. transfection

With plasmid, the sgRNA1 of expression Cas9_ANTXR1Or sgRNA2_ANTXR1Or pgRNA_ANTXR1And their corresponding confessions Body cotransfection HeLa_OCCell individually uses linear donor dna (Donor as control_ANTXR1-sg1、Donor_ANTXR1-sg2With Donor_ANTXR1-pg) transfection HeLa_OCCell, and puromycin is added and carries out resistance screening, it is dyed with MTT, as a result such as Fig. 5 b institute Show.

Similar with the result in HeLa cell, only donor obtains a large amount of puro+ clone plus sgRNA.

4. the verifying of gene knockout efficiency

In addition with the plasmid and sgRNA1 of expression Cas9_ANTXR1Or sgRNA2_ANTXR1Or pgRNA_ANTXR1, add or they be not added Corresponding donor, cotransfection HeLa_OCCell is screened with puromycin (1 μ g/ml).Screening is handled with PA/LFnDTA to obtain Cell, treated that image is as shown in Figure 5 c with PA/LFnDTA for different cells.Have by calculating in puro+ mictium There is the percentage of the cell of the toxin resistance to determine that ANTXR1 knocks out efficiency, as fig 5d, compared with sgRNA is used alone, The use of linear donor dna substantially increases ANTXR1 gene knockout efficiency.

To the single clone of puro+, to donor Donor_ANTXR1-s_g1Integration site on ANTXR1 gene carries out PCR and tests Card, it is found that the major part of puro+ is cloned in sgRNA target site and contains donor insert (Fig. 5 e and Fig. 5 f), most of to carry The cell of donor fragment is real gene knockout clone (Fig. 5 f).

(length is corresponding to open country by the PCR fragment of right~500bp (length corresponds to wild type ANTXR1 gene) and~1.8kb Raw type ANTXR1 gene adds donor insert) PCR fragment carry out gene order-checking, the results are shown in Table 4.

The gene order-checking result of the PCR fragment of 4~500bp of table and the PCR fragment of~1.8kb

Target site: ANTXR1 (Chr2, HeLa_oc)

From PCR verification result (Fig. 5 e) and sequencing result (table 4), it can be seen that, most of clone contains only a donor and inserts Enter.But in donor positive colony, most allele are edited and (are mutated in other words) in target site, still The efficiency that individual sgRNA generates insertion or deletion mutation (indels) without using donor enrichment is very low.This It was found that obviously proving that donor insertion is closely related with the effect of sgRNA or pgRNA.

5. influence of the donor to the undershooting-effect of CRISPR/Cas system

In order to check whether the use of external donor will affect the off-target effects of CRISPR/Cas system, pass through Splinkerette PCR analyzes the integration site [35-37] for finding full-length genome.

Verified after puromycin selection in the present embodiment by splinkerette PCR analysis in single clone and The insertion missed the target in cell mixing clone.If there is the insertion of correct donor on ANTXR1 gene, with primer Splink2/ R1 and Splink2/R2 amplification can generate the product of 711- and 927-bp respectively (referring to Fig. 6 a).

In order to carry out splinkerette PCR analysis, we have been randomly choosed in HeLa_OCTarget ANTXR1's in cell 10 single clones and 4 puro+ mixing clones with donor insertion.According to splinkerette PCR result (Fig. 6 b), Similar with the clone that those are not transfected with donor, the single clone or mictium being enriched with by donor not can be detected Undershooting-effect.

Embodiment 5 is enriched with HeLa using linear donor dna_OCKnockout event in cell on HBEGF gene

The design of 1.sgRNA and the building of linear donor dna

Design two targeting HeLa_OCSgRNA (the sgRNA1 of HBEGF gene in cell_HBEGFAnd sgRNA2_HBEGF) and construct Three linear donor dna (Donor_HBEGF-sg1、Donor_HBEGF-sg2And Donor_HBEGF-pg), see Fig. 7 a.Wherein sgRNA1_HBEGFNeedle Pair target sequence in the present embodiment be referred to as sg1, sgRNA2_HBEGFFor target sequence in the present embodiment be referred to as sg2.

Donor_HBEGF-sg2The end from 5' to 3' respectively include: the protection sequence of 20bp, sg2, reversed terminator codon, CMV are opened The puromycin resistance gene of mover driving, positive terminator codon, the protection sequence of 20bp.

Donor_HBEGF-pgThe end from 5' to 3' respectively include: the protection sequence of 20bp, sg1, reversed terminator codon, CMV are opened The puromycin resistance gene of mover driving, positive terminator codon, the protection sequence of sg2,20bp.

3. transfection

With plasmid, the sgRNA1 of expression Cas9_HBEGFOr sgRNA2_HBEGFOr pgRNA_HBEGFAnd their corresponding donors Cotransfection HeLa_OCCell individually uses linear donor dna (Donor as control_HBEGF-sg1、Donor_HBEGF-sg2With Donor_HBEGF-pg) transfection HeLa_OCCell, and puromycin is added and carries out resistance screening, as a result as shown in Figure 7b.

4. the verifying of gene knockout efficiency

To the single clone of puro+, to donor Donor_HBEGF-sg1Integration site on HBEGF gene carries out PCR and tests It demonstrate,proves, L2/R2 primer sequence used in PCR amplification is as shown in table 5.It was found that being cloned in sgRNA targeting from the major part of puro+ Site is contained donor insert (Fig. 7 c and Fig. 7 d), and most of cell for carrying donor fragment is real gene knockout clone (Fig. 7 d).

Table 5 is for expanding HeLa_OCThe primer in the site HBEGF that linear donor is integrated in cell

Primer pair	Sequence
		L2/R2	5'-GCCGCTTCGAAAGTGACTGG-3'/5'-GATCCCCCAGTGCCCATCAG-3'

Embodiment 6 is using linear donor dna in HeLa_OCDual-gene knockout is carried out in cell

The design of 1.sgRNA

Selected HeLa_OCTwo target genes in cell: PSEN1 and PSEN2, design target the two of the two target genes respectively A sgRNA, by T7E1 measurement verifying, they generate the efficiency of Indels on target site, and the results are shown in Table 6.Wherein sgRNA_PSEN1For target sequence in the present embodiment be referred to as sg_PSEN1, sgRNA_PSEN2For target sequence in the present embodiment Referred to as sg_PSEN2。

Table 6 targets HeLa_OCThe sgRNA of PSEN1 and PSEN2 gene in cell

2. the building of linear donor dna

Construct two kinds of donor, there are two individual donor (Donor for a kind of tool_PSEN1+Donor_PSEN2), each confession Body has the target sequence of corresponding sgRNA, another donor (Donor_PSEN) at both ends two sgRNA target sequences are respectively provided with, such as Shown in Fig. 8 a.Donor_PSEN1Or Donor_PSEN2There is sgRNA in its end 5'_PSEN1Or sgRNA_PSEN2Cleavage site. Donor_PSEN1+PSEN2There is sgRNA at the end 5'_PSEN1Cleavage site has sgRNA at the end 3'_PSEN2Cleavage site.Wherein:

Donor_PSEN1The end from 5' to 3' respectively include: the protection sequence of 20bp, sg_PSEN1, reversed terminator codon, CMV opens The puromycin resistance gene of mover driving, positive terminator codon, the protection sequence of 20bp.

Donor_PSEN2The end from 5' to 3' respectively include: the protection sequence of 20bp, sg_PSEN2, reversed terminator codon, CMV opens The puromycin resistance gene of mover driving, positive terminator codon, the protection sequence of 20bp.

Donor_PSENThe end from 5' to 3' respectively include: the protection sequence of 20bp, sg_PSEN1, reversed terminator codon, CMV opens The puromycin resistance gene of mover driving, positive terminator codon, sg_PSEN2, the protection sequence of 20bp.

3, the verifying of transfection and gene knockout efficiency

With plasmid, the sgRNA of expression Cas9_PSEN1Or sgRNA_PSEN2Cotransfection HeLa_OCCell, or with expression Cas9's Plasmid, pgRNA_PSENCotransfection HeLa_OCCell, to generate indels on the specific site of PSEN1 and PSEN2 gene.It is right Indels generation efficiency carries out T7E1 analysis [26] (primer used is shown in Table 7), as a result as illustrated in fig. 9, their cotransfection All only show active.

With plasmid, the pgRNA of expression Cas9_PSENAnd Donor_PSENCotransfection HeLa_OCCell, or with expression Cas9's Plasmid, pgRNA_PSENAnd Donor_PSEN1+Donor_PSEN2Cotransfection HeLa_OCCell, and puromycin is added and carries out resistance sieve Choosing can obtain puro+ clone (see Fig. 9 b).Similar with the result of preceding embodiment, donor obtains a large amount of plus pgRNA Puro+ clone.

For every kind of transfection results, the single clone of puro+ is taken, to donor Donor_PSENAnd Donor_PSEN1+Donor_PSEN2 Integration site on PSEN1 and PSEN2 gene carries out PCR verifying, and primer used in PCR amplification is as shown in table 7, Middle L3/R3 is used to expand the integration site on PSEN1, and L4/R4 is used to expand the integration site on PSEN2, PCR verification result As is shown in fig. 9 c, on two genes all containing donor insertion clone marked with box, selection clone 1 and clone 2 further into The analysis of row gene order-checking, two are cloned the destruction (Fig. 8 b and Fig. 8 c) for all showing PSEN1 and PSEN2.

Table 7 is for expanding HeLa_OCThe primer in the site PSEN1 and PSEN2 that linear donor is integrated in cell

Primer pair	Sequence
		L3/R3	5'-TGGTGTCTCAGGCGGTTCTA-3'/5'-TGAACTATGAGGCGCTGCAC-3'
L4/R4	5'-TGACTTTCGTGGCTATGCGT-3'/5'-CTAGCACCCAGGCATCCAAA-3'

Embodiment 7 is using linear donor dna in HeLa_OCPolygenes knockout is carried out in cell

1. the selection of target gene and the design of sgRNA

Select HeLa_OCHSPA gene family in cell, the gene family there are five gene, HSAPA1A, HSPA1B, HSBA1L, HSPA6 and HSPA2, they have homology.It designs while targeting HSAPA1A, HSPA1B and HSBA1L sgRNA_HSPA, the corresponding sequence on the target sequence and HSPA6 of the sgRNA has a mispairing, and there are two mispairing with HSPA2.Such as figure Shown in 8d.

2. the building of linear donor dna

Construct linear donor Donor_HSPA, the end from 5' to 3' respectively include: the protection sequence of 20bp, sg_HSPA, reversed to terminate Codon, the puromycin resistance gene of CMV promoter driving, positive terminator codon, the protection sequence (Fig. 8 d) of 20bp.

3. the verifying of transfection and gene knockout efficiency

With the plasmid and sgRNA of expression Cas9_HSPA, add or be not added its corresponding donor Donor_HSPA, cotransfection HeLa_OCCarefully Born of the same parents cause indels, and carry out resistance screening by puromycin,.The group and expression puromycin-resistant of donor is wherein not added The plasmid co-transfection of gene.By T7E1 measurement (primer used is shown in Table 8) evaluation institute there are five gene indels efficiency, As a result as figure 8 e shows, with individual sgRNA_HSPAIt compares, donor HSPA is used to mention in the mutation rate in the site HSPA1A About 5.5 times high, the mutation rate in the site HSPA1B improves about 6.1 times, and the mutation rate in the site HSPA1L improves about 3.4 times, the mutation rate in the site HSPA6 improves about 6.6 times, it is interesting that supplying whether or not using on HSPA2 gene Body does not all detect indels, shows that two mispairing destroy sgRNA completely_HSPAIdentification, moreover, importantly, The risk for not increasing undershooting-effect is selected using donor.

To the target region in HSPA family gene in the cell mixing with donor cotransfection and obtained with donor cotransfection It is sequenced, the results are shown in Figure 10, the results showed that no matter on HSPA family gene site whether have donor transfection, cell It is all consistent with the result of T7E1 measurement to mix sequencing result.

It is worth noting that, T7E1 measurement proves the selected highly enriched cell for carrying target mutation of group, with biography The method without using donor of system is compared, and concentration coefficient is about 753 (5.5*6.1*3.4*6.6).Not in view of this calculating Consider the gene with donor insertion, actual efficiency improves even higher.

To the single clone with puromycin-resistant, PCR verifying is carried out on five target sites, for expanding all five The specific primer (L5/R5, L6/R6, L7/R7, L8/R8, L9/R9) that the target site of a gene uses is listed in Table 8.As a result As shown in figures 11a and 11b.Six clones of the selection wherein at least two target genes with donor insertion (use side in figure It is 1-6 that frame, which is identified and numbered respectively) genomic sequence analysis is carried out, as a result as shown in figure 11b, clone 3 is in four genes With modification in corresponding site: there is frameshift mutation on HSPA1A, HSPA1B and HSPA1L, generate complete knockout, There are two be mutated (Fig. 8 f) in frame for tool on HSPA6.

Table 8 is for expanding HeLa_OCThe primer of five gene target sites of HSPA family in cell

Primer pair	Sequence
		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'

Embodiment 8 utilizes the knockout on CSPG4 gene in the linear donor dna enrichment SC-8 cell containing general sgRNA Event

1, the screening of general sgRNA

Following 10 sgRNA candidate sequence as a filter is selected, as shown in the table:

2. the building of linear donor dna

It is based respectively on above-mentioned 10 kinds general sgRNA and constructs 10 kinds of linear donor dna (Donor_{SgRNA_Universal_1~10-puro})。

These linear donor dnas end from 5' to 3' respectively include: the protection sequence of 20bp, sgRNA_{Universal_1~10}Target Sequence, reversed terminator codon, the puromycin resistance gene of CMV promoter driving, positive terminator codon, the guarantor of 20bp Protect sequence.

3. the building for sgRNA plasmid of connecting

It is based respectively on above-mentioned 10 kinds of linear donors and constructs 10 kinds of series connection sgRNA plasmid (Plasmid_{PgRNA_Universal_1~10}), Its structure is referring to Figure 12.

Concatenated two sgRNA are respectively sgRNA_CSPG4And sgRNA_{Universal_1~10}.Wherein sgRNA_CSPG4Target TcdB poison The receptor CSPG4, sgRNA of element_{Universal_1~10}Target corresponding donor dna (Donor_{SgRNA_Universal_1~10-puro}) in target Sequence.

4, it transfects

Cell line used in transfection experiment is to stablize the cell line (SC-8) of expression Cas9, with ten kinds of Plasmid series Plasmid_{PgRNA_Universal_1~10}With corresponding donor dna (Donor_{SgRNA_Universal_1~10-puro}) cotransfection SC-8 cell, make For control, independent ten kinds of linear donor dna (Donor_{SgRNA_Universal_1~10-puro}) transfection SC-8 cell.And purine is added Mycin carries out resistance screening, obtains mictium (pooled population), the selection result is as shown in the table.

According to the above results, sgRNA_{Universal_1}、sgRNA_{Universal_3}、sgRNA_{Universal_6}And sgRNA_{Universal_9}Four Kind sgRNA effect is preferable, therefore subsequent experimental uses the corresponding mixing bacterium colony of these four sgRNA as experimental subjects.

4. the verifying of gene knockout efficiency

TcdB toxin is added in four kinds of mixing bacterium colonies to be screened, cell survival is observed after 23 hours, is tested As a result as shown in figure 13.As can be seen that sgRNA is added after being added TcdB toxin 23 hours_{Universal_1}And sgRNA_{Universal_9} The corresponding cell survival rate of experimental group is apparently higher than remaining two groups, illustrates sgRNA_{Universal_1}And sgRNA_{Universal_9}Gene It knocks out more efficient.

Material used in above embodiments 1-7 and method are as follows:

Cell culture and transfection

HeLa, HeLaOC and HEK293T cell are maintained at added with 10% fetal calf serum (FBS, Lanzhou lark biology skill Art Co., Ltd, Lanzhou, China) Dulbecco ' s modified Eagle ' s culture medium (DMEM, 10-013-CV, Corning, Tewksbury, MA, USA) in, temperature is 37 DEG C, is passed through 5%CO₂.In order to be transfected, all cells are connect Kind in 6 orifice plates, and with X-tremeGENE HP (06366546001, Roche, Mannheim, Germany) transfection, according to Supplier's illustrates to carry out.Briefly, 2 μ g DNA and 4 μ l X-tremeGENE HP are added to 200 μ l Opti-MEM I Reduced Serum Medium(31985088,Thermo Fisher Scientific,Grand Island,NY,USA) In.Mixture is incubated 15 minutes in room temperature, is then added in cell.

Express the clone of the plasmid of gRNA

For expressing the plasmid of sgRNA, the oligonucleotides (being shown in Table 9) of individually designed each sgRNA coded sequence, and carry out It synthesizes (Beijing Bioisystech Co., Ltd, farsighted Boxing section).

The primer that table 9 is constructed for sgRNA or pgRNA

It is 10 μM that oligonucleotides, which is dissolved to concentration, with 1 × TE, with TransTaq HiFi Buffer II (K10222, north Jing Quanshijin Bioisystech Co., Ltd) it is mixed into oligonucleotides, it is heated to 95 DEG C and is kept for 3 minutes, then slowly cooled to 4℃.In 37 DEG C of oligonucleotides by these annealing to phosphorylation 30 minutes, after being heated and inactivated, " Golden Gate " method is used Product is connected in sgRNA skeleton carrier.For expressing the plasmid of pgRNA, with containing there are two the primers of gRNA coded sequence Expand gRNA scaffold sequence and U6 promoter (table 5), then purified pcr product and use " Golden Gate " method connect It is connected in sgRNA skeleton carrier.Compared with the sgRNA skeleton carrier [17] being previously reported, sgRNA skeleton carrier of the invention SgRNA skeleton is modified [38], and with mCherry coded sequence instead of EGFP sequence.

T7E1 measurement

Genome is extracted using DNeasy Blood&Tissue kit (69504, Qiagen, Hilden, Germany) DNA carries out PCR amplification to the genome area containing gRNA target sequence.Primer sequence used in measurement such as table 2, table 5, table 7, shown in table 8.Used primer sequence such as takes the PCR product and 10 × NEB of 300-500ng in 50 μ l systems Buffer2 mixing, 95 DEG C heating 3 minutes after be slowly cooled to room temperature.Products therefrom is added 0.5 μ l T7E1 and incubates at 37 DEG C 15min, carries out agarose gel electrophoresis, and electrophoretogram analyzes the efficiency that band is cut, instruction with Image J image analysis software The efficiency of sgRNA generation Indels.

Linear donor building

The sequence of the pre-generated donor containing the CMV puromycin resistance gene driven or EGFP gene and terminator codon Column, and be cloned into pEASY-T5-Zero cloning vector (CT501-02, Beijing Quanshijin Biotechnology Co., Ltd), as Common template.Using the primer amplification template containing sgRNA cutting target site and protection sequence, primer sequence is as shown in table 10.

Table 10 is used for the primer of linear donor building

Donor insertion and cell selection based on NHEJ

In HeLa_OCIn cell, cell is transfected with the 1 μ g linear donor PCR product purified and 1 μ g sgRNA/pgRNA, is turned Two weeks after dye, cell is handled with the puromycin of 1 μ g/ml.In HeLa and HEK293T cell, with 1 μ g donor and 0.5 μ g SgRNA/pgRNA and 0.5 μ g Cas9 plasmid-transfected cells.Then two weeks puromycins with 1 μ g/ml are handled after transfection Cell, or the EGFP positive is determined by the cell sorting (FACS) of fluorescence-activation, this depends on using which type of confession Body.

Splinkerette PCR

(Potter, C.J.&Luo, L.Splinkerette PCR previously had been reported in Splinkerette PCR method for mapping transposable elements in Drosophila.PLoS One5,e10168(2010)；Uren, A.G.et al.A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites.Nat Protoc4,789-798(2009)；Yin,B.& Largaespada,D.A.PCR-based procedures to isolate insertion sites of DNA elements.Biotechniques43,79-84(2007)).Used primer and connector (adaptor) sequence such as table 11 It is shown.

Table 11 is used for the primer of Splinkerette PCR

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Claims

1. universal donor construct, the universal donor construct is linear donor dna or can generate line by cutting in cell Property donor dna, the linear donor dna successively includes from centre to both ends: expression cassette；Positioned at the end expression cassette 5' by reversed whole Only password molecular short sequence extension and being prolonged by the molecular short sequence of positive termination codon positioned at the end expression cassette 3' It stretches；General target sequence positioned at the end 5' and/or the end 3', the general target sequence contain the target position that can be cut by Cas9 nuclease Point；Protection sequence positioned at both ends；

Wherein the expression cassette includes the marker gene driven by promoter；

2. the universal donor construct of claim 1, is linear donor dna, preferably double stranded donor dna.

Preferably, the linear donor dna only has the general target sequence or the linear donor at the end 5' or the end 3' DNA is respectively provided with the general target sequence at both ends.

It is highly preferred that wherein the marker gene is antibiotics resistance gene or fluorescence protein gene.

3. the universal donor construct of any one of claim 1-2, wherein the length of the protection sequence is 5-30bp, most preferably 20bp。

Preferably, the general target sequence contain 5'-GTACGGGGCGATCATCCACA-3' or

5'-AATCGACTCGAACTTCGTGT-3'。

4. the method for generating gene knockout in cell, the described method comprises the following steps:

(1) it is introduced into cell:

(a) Cas9 nuclease；

(b) gRNA of specific target sequence in cellular genome is identified；

(c) universal donor construct, wherein the universal donor construct is linear donor dna or can be produced in cell by cutting Raw linear donor dna, the linear donor dna successively includes from centre to both ends: expression cassette；Positioned at the end expression cassette 5' by anti- Extend and positioned at the end expression cassette 3' to the molecular short sequence of termination codon by the molecular short sequence of positive termination codon Column extend；General target sequence positioned at the end 5' and/or the end 3', the general target sequence contain the target that can be cut by Cas9 nuclease Site；Protection sequence positioned at both ends；

Wherein the expression cassette includes the marker gene driven by promoter；

(d) gRNA for the general target sequence for including in linear donor dna is identified；

(3) cell of the marker representation positive is screened.

5. method for claim 4, wherein the universal donor construct is linear donor dna, preferably double stranded donor DNA。

It is highly preferred that the linear donor dna only has the general target sequence or the linear donor at the end 5' or the end 3' DNA is respectively provided with the general target sequence at both ends.

6. the method for any one of claim 4-5, wherein the gRNA of specific target sequence is a kind of in the identification cellular genome The gRNA of different target sequences in gRNA or more than one identification cellular genome.

Preferably, the gRNA of specific target sequence is sgRNA and/or the identification linear donor in the identification cellular genome The gRNA for the general target sequence for including in DNA is sgRNA.

It is highly preferred that wherein in the identification cellular genome in the sgRNA and the identification linear donor dna of specific target sequence The sgRNA for the general target sequence for including is located in identical carrier；Or

In the identification cellular genome sgRNA of specific target sequence and it is described identification linear donor dna in include general target The sgRNA of sequence is located in different carriers.

7. the method for any one of claim 4-6, wherein the marker gene is antibiotics resistance gene or fluorescin base Cause.

Preferably, wherein screening cell by drug resistance, or cell is screened by FACS method.

Preferably, wherein the length of the protection sequence is 5-30bp, most preferably 20bp.

It is highly preferred that wherein the general target sequence contain 5'-GTACGGGGCGATCATCCACA-3' or

5'-AATCGACTCGAACTTCGTGT-3'。

8. it is used for the system or kit of gene knockout, including:

(1) Cas9 nuclease or the carrier or cell of Cas9 nuclease can be expressed；

(2) gRNA of specific target sequence in cellular genome is identified；

(3) universal donor construct, wherein the universal donor construct is linear donor dna or can be produced in cell by cutting Raw linear donor dna, the linear donor dna successively includes from centre to both ends: expression cassette；Positioned at the end expression cassette 5' by anti- Extend and positioned at the end expression cassette 3' to the molecular short sequence of termination codon by the molecular short sequence of positive termination codon Column extend；General target sequence positioned at the end 5' and/or the end 3', the general target sequence contain the target that can be cut by Cas9 nuclease Site；Protection sequence positioned at both ends；

Wherein the expression cassette includes the marker gene driven by promoter；

9. the system or kit of claim 8, wherein the universal donor construct is linear donor dna, preferably double-strand Linear donor dna.

Preferably, wherein the linear donor dna only has the general target sequence or the linear confession at the end 5' or the end 3' Body DNA is respectively provided with the general target sequence at both ends.

It is highly preferred that wherein the gRNA of specific target sequence is a kind of gRNA in the identification cellular genome, or it is more than one The gRNA of different target sequences in the identification cellular genome of kind.

It is more preferred still that wherein the gRNA of specific target sequence is sgRNA and/or the identification in the identification cellular genome The gRNA for the general target sequence for including in linear donor dna is sgRNA.

It is particularly preferred that wherein it is described identification cellular genome in specific target sequence sgRNA and the identification linear donor dna In include the sgRNA of general target sequence be located in identical carrier；Or

10. the system or kit of claim 9, wherein the marker gene is antibiotics resistance gene or fluorescin base Cause.

It is further preferred that wherein the general target sequence contain 5'-GTACGGGGCGATCATCCACA-3' or

5'-AATCGACTCGAACTTCGTGT-3'。