CN109371167A

CN109371167A - Genetic elements and the application of frameshift mutation are generated for detecting CRISPR/Cas9 gene editing system cutting gene

Info

Publication number: CN109371167A
Application number: CN201811320658.5A
Authority: CN
Inventors: 鲁文静; 兰峰; 张治宇
Original assignee: BEIJING CELLAPY BIOTECHNOLOGY Co Ltd
Current assignee: BEIJING CELLAPY BIOTECHNOLOGY Co Ltd
Priority date: 2018-11-07
Filing date: 2018-11-07
Publication date: 2019-02-22

Abstract

The invention discloses a kind of Genetic elements and applications that frameshift mutation is generated for detecting CRISPR/Cas9 gene editing system cutting gene.The Genetic elements include the promoter effectively connected, the first reporter gene, REST gene, SV40pA gene, S silencer, the second reporter gene and terminator, wherein, it can be inserted between promoter and the first reporter gene by cutting target dna sequence, first reporter gene is different from the second reporter gene, and the expression of REST gene is able to suppress the expression of the second reporter gene behind S silencer.The Genetic elements that frameshift mutation is generated for detecting CRISPR/Cas9 gene editing system cutting gene can be used as core element and be connected in carrier, it can be used to the cutting efficiency in living cells identification gRNA intracellular to genome, be the fluorescence phenotype and ease for operation of instant fluorescence phenotype, high stability when the detection is with generation frameshift mutation.

Description

Frameshift mutation is generated for detecting CRISPR/Cas9 gene editing system cutting gene Genetic elements and application

Technical field

The present invention relates to the clone technologies and field of molecular detection in genetic engineering, in particular to a kind of use Genetic elements and the application of frameshift mutation are generated in detection CRISPR/Cas9 gene editing system cutting gene.

Background technique

The short palindrome repetitive sequence in the interval of regular cluster (Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR) it is one of most of bacteriums and archeobacteria acquired immunity mode.2012, Jinek etc. operates genomic DNA using CRISPR/Cas9 gene editing system, and confirms that this system can be Active somatic cell realizes the gene editing that RNA is mediated.So far, CRISPR/Cas9 gene editing system is widely used in target gene Editor.Especially in inductivity versatile stem cell (Induced Pluripotent Stem cells, iPS cells) or embryo Application in tire stem cell (Embryonic Stem Cell, ESCs), uses CRISPR/Cas9 gene editing system modification base Because the cell line for organizing obtained can imitate gene patient, the research and development for purpose new drug, specific drug；Or inactivation specific mutation Gene achievees the purpose that gene editing treats disease.On the other hand, since CRISPR/Cas9 gene editing system is that RNA is mediated , thus gRNA whether have single-minded cleavage site be it is most crucial, restrict CRISPR/Cas9 gene editing system and use Factor is one of the key element for determining foregoing purpose realization.Therefore, gRNA design quality determines what target gene group was cut Efficiency；The gRNA of specificity can reduce the probability that misses the target, and improve the accuracy of gene editing, avoid the editor of extra false.Institute To need a kind of detection method to carry out gRNA designed by Rapid identification and generate frameshift mutation to the cutting efficiency of genome and editor How genome base sequence afterwards is distributed.

Summary of the invention

The present invention is intended to provide a kind of generate frameshift mutation for detecting CRISPR/Cas9 gene editing system cutting gene Genetic elements and application, with provide it is a kind of quickly, can be in living cells identification gRNA intracellular to the cutting efficiency of genome Method.

To achieve the goals above, according to an aspect of the invention, there is provided it is a kind of for detecting CRISPR/Cas9 base Because editing system cutting gene generates the Genetic elements (CRISPR/Cas9Indel Assay system, CIAs) of frameshift mutation. CIAs Genetic elements include the promoter effectively connected, the first reporter gene, REST gene, SV40pA gene, S silencer, Two reporter genes and terminator, wherein it is can be inserted between promoter and the first reporter gene by cutting target dna sequence, First reporter gene is different from the second reporter gene, and the expression of REST gene is able to suppress the second reporter gene behind silencer Expression.

Further, Genetic elements further include the enhancer effectively connected.

Further, enhancer is cmv enhancer.

Further, the first reporter gene is red fluorescent protein gene, and the second reporter gene is green fluorescent protein base Cause.

Further, promoter is EF1 promoter, and terminator is beta-globin polyadenylic acid.

Further, Genetic elements contain double enzyme site, and double enzyme site is located between promoter and the first report, quilt Cutting target dna sequence is connected between double enzyme site.

Further, double enzyme site is Xba I and Kpn I restriction enzyme site.

Further, REST gene and the amplification of S silencer are in source of people H9 cell line.

Further, Genetic elements have the nucleotide sequence as shown in SEQ ID NO:1.

According to another aspect of the present invention, it provides a kind of for detecting CRISPR/Cas9 gene editing system cutting base Because generating the carrier of frameshift mutation.The carrier includes any of the above-described kind of Genetic elements.

Further, carrier is pUC serial carrier, pEASY, pBlueScriptII or pBR322.

According to another aspect of the present invention, a kind of host cell is provided.The host cell includes any of the above-described kind and is used for Detect the carrier that CRISPR/Cas9 gene editing system cutting gene generates frameshift mutation.

Further, host cell is Escherichia coli.

In accordance with a further aspect of the present invention, a kind of detection CRISPR/Cas9 gene editing system cutting gene production is provided The method of raw frameshift mutation.This method is to cut base for detecting CRISPR/Cas9 gene editing system using any of the above-described kind Carrier because generating frameshift mutation is detected.

Further, comprising the following steps: then S1 turns by any of the above-described kind of carrier insertion by cutting target dna sequence Dye is transferred to the carrier (DNA sequence dna comprising expressing Cas9 albumen) for being mounted with gRNA into cell line；S2, observation first The expression of reporter gene and the second reporter gene judges according to the expression of the first reporter gene and the second reporter gene Whether gRNA produces frameshift mutation to the editor of target dna sequence.

Further, further includes: when the second reporter gene normal expression, be judged as CRISPR/Cas9 gene editing system System cutting gene produces frameshift mutation, and expands target dna sequence and be sequenced.

Further, cell line is HEK293 cell line.

The Genetic elements of frameshift mutation are generated (referred to as detecting CRISPR/Cas9 gene editing system cutting gene CIAs it) can be used as core element to be connected in carrier, can be used to imitate the cutting of genome in living cells identification gRNA intracellular Rate, the detection have generate frameshift mutation when be instant fluorescence phenotype, high stability fluorescence phenotype and ease for operation, and The vector construction has the remarkable advantage of the genetic manipulations such as simple, quick, time saving, laborsaving.

Detailed description of the invention

The accompanying drawings constituting a part of this application is used to provide further understanding of the present invention, and of the invention shows Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.In the accompanying drawings:

Fig. 1 shows Genetic elements CIAs composition schematic diagram.；

Fig. 2 a and 2b are respectively the plasmid map of the CIAs-pUC19 that Genetic elements CIAs of the invention is loaded and pCIAs.

Fig. 3 a and 3b points of target-pCIAs and target-1-pCIAs to load target DNA sequence.

Fig. 4 a, 4b, 4c and 4d are respectively the plasmid map for loading gRNA sequence.

Fig. 5 a, 5b, 5c and 5d are respectively that the light microscopic and fluorescence photo of bacterial strain to be edited are edited and do not had to target DNA.

Specific embodiment

It should be noted that in the absence of conflict, the features in the embodiments and the embodiments of the present application can phase Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments.

For some technical problems existing in the prior art, the present invention provides a kind of detection CRISPR/Cas9 gene volume The system of collecting cutting gene generates Genetic elements, carrier, host cell and the method for frameshift mutation.

A kind of typical embodiment according to the present invention provides a kind of for detecting CRISPR/Cas9 gene editing system Cut the Genetic elements that gene generates frameshift mutation.The Genetic elements include the promoter effectively connected, the first reporter gene, REST gene, SV40pA gene, S silencer, the second reporter gene and terminator, wherein promoter and the first reporter gene base It is can be inserted into because between by cutting target dna sequence, the first reporter gene is different from the second reporter gene, the expression energy of REST gene Enough inhibit the expression of the second reporter gene behind silencer.

" effectively connection " the i.e. conventional sense of field of biotechnology in the present invention, that is, the Genetic elements after connecting can rise To its scheduled effect.

Target dna sequence is connected in said gene element, and gRNA/Cas9 complex cuts target dna sequence and generates When frameshift mutation, the first reporter gene and REST gene can also generate frameshift mutation, be that cannot express correct first report base Because of-REST fusion protein, the albumen of the first reporter gene can not be as generated, the second report behind S silencer can not be inhibited The expression of gene.At this moment, the albumen of the second reporter gene can normal expression, can make host cell line generate second report The signal of gene.

Target dna sequence is connected in Genetic elements CIAs, and gRNA/Cas9 complex cutting target dna sequence does not produce When raw frameshift mutation, the first reporter gene and the correct first reporter gene-REST fusion protein of REST gene expression are as produced The albumen of raw first reporter gene, while S silencer inhibits the expression of the second reporter gene below.At this moment, the first reporter gene Can normal expression, and the second reporter gene is not expressed, so the signal of the first reporter gene.

When the first reporter gene is red fluorescent protein gene, and the second reporter gene is green fluorescence protein gene, mesh Mark DNA sequence dna is connected in Genetic elements CIAs, when gRNA/Cas9 complex cuts target dna sequence generation frameshift mutation, Genetic elements red fluorescent protein and REST gene can also generate frameshift mutation, be that cannot express correct red fluorescence-REST Fusion protein as can not generate red fluorescent protein and not inhibit the expression of gene green fluorescent protein behind silencer.This When, green fluorescent protein can normal expression, can make host generate green fluorescence.Target dna sequence is connected to gene In element CIAs, when gRNA/Cas9 complex cutting target dna sequence does not generate frameshift mutation, Genetic elements red fluorescence egg The white and correct red fluorescence-REST fusion protein of REST gene expression, as generation red fluorescent protein and inhibition silencer The expression of gene green fluorescent protein below.At this moment, red fluorescent protein can normal expression, and green fluorescence is not table It reaches, so host generates red fluorescence.

The sequence that said gene element CIAs is included by gene chemical synthesis or can be amplified from and contain this Genetic elements Target nucleic acid sequence obtain, and linked together by the method for fusion DNA vaccine or homologous recombination.Composition sequence separate sources, It can be applied to different cell lines.Wherein, a kind of typical embodiment, REST gene and S silencer expand according to the present invention In source of people iPS or H9 cell line, it is more suitable for the gene frameshift mutation detection of human archeocyte system.

Preferably, Genetic elements further include the enhancer effectively connected, and preferred enhancer is cmv enhancer.

A kind of typical embodiment according to the present invention, promoter are EF1 promoter, and terminator is the poly- adenosine of beta-globin Acid.

Convenient for target dna sequence, Genetic elements CIAs contains double enzyme site, and double enzyme site is located at promoter and first Between report, it is connected between double enzyme site by cutting target dna sequence.Preferably, double enzyme site is Xba I and Kpn I restriction enzyme site.

Preferably, Genetic elements CIAs has the nucleotide sequence as shown in SEQ ID NO:1.

SEQ ID NO:1

TGTAAAACGACGGCCAGTGAATTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGG TCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGT ACATCTACGTATTAGTCATCGCTATTACCATGGAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATT ACAAAAATTCAAAATTTTATCGATACTAGTAAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA TCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAA ACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGT CGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCT TCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTG CCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTG GAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTT TCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACATGTCTAGANNNNNNNNNNNNNNNNNNNNNNN NNNNNNNGGTACCGTGTCTAAGGGCGAAGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACC GTGAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGACCATGAGAATCA AGGTGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCAGCTTCATGTACGGCAGCAAAACCTT CATCAACCACACCCAGGGCATCCCCGACTTCTTTAAGCAGTCCTTCCCTGAGGGCTTCACATGGGAGAGAGTCACC ACATACGAAGACGGGGGCGTGCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCA AGATCAGAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCTCCACCGA GATGCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAAGCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTG ATCTGCAACTTGAAGACCACATACAGATCCAAGAAACCCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGG ACAGAAGACTGGAAAGAATCAAGGAGGCCGACAAAGAAACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGATA CTGCGACCTCCCTAGCAAACTGGGGCACAAACTTAATTAAGCCACCCAGGTAATGGGGCAGTCTTCTGGAGGAGGA GGGCTGTTTACCAGCAGTGGCAACATTGGAATGGCCCTGCCTAACGACATGTATGACTTGCATGACCTTTCCAAAG CTGAACTGGCCGCACCTCAGCTTATTATGCTGGCAAATGTGGCCTTAACTGGGGAAGTAAATGGCAGCTGCTGTGA TTACCTGGTCGGTGAAGAAAGACAGATGGCAGAACTGATGCCGGTTGGGGATAACAACTTTTCAGATAGTGAAGAA GGAGAAGGACTTGAAGAGTCTGCTGATATAAAAGGTGAACCTCATGGACTGGAAAACATGGAACTGAGAAGTTTGG AACTCAGCGTCGTAGAACCTCAGCCTGTATTTGAGGCATCAGGTGCTCCAGATATTTACAGTTCAAATAAAGATCT TCCCCCTGAAACACCTGGAGCGGAGGACAAAGGCAAGAGCTCGAAGACCAAACCCTTTCGCTGTAAGCCATGCCAA TATGAAGCAGAATCTGAAGAACAGTTTGTGCATCACATCAGAGTTCACAGTGCTAAGAAATTTTTTGTGGAAGAGA GTGCAGAGAAGCAGGCAAAAGCCAGGGAATCTGGCTCTTCCACTGCAGAAGAGGGAGATTTCTCCAAGGGCCCCAT TCGCTGTGACCGCTGCGGCTACAATACTAATCGATATGATCACTATACAGCACACCTGAAACACCACACCAGAGCT GGGGATAATGAGCGAGTCTACAAGTGTATCATTTGCACATACACAACAGTGAGCGAGTATCACTGGAGGAAACATT TAAGAAACCATTTTCCAAGGAAAGTATACACATGTGGAAAATGCAACTATTTTTCAGACAGAAAAAACAATTATGT TCAGCATGTTAGAACTCATACAGGAGAACGCCCATATAAATGTGAACTTTGTCCTTACTCAAGTTCTCAGAAGACT CATCTAACTAGACATATGCGTACTCATTCAGGTGAGAAGCCATTTAAATGTGATCAGTGCAGTTATGTGGCCTCTA ATCAACATGAAGTAACCCGCCATGCAAGACAGGTTCACAATGGGCCTAAACCTCTTAATTGCCCACACTGTGATTA CAAAACAGCAGATAGAAGCAACTTCAAAAAACATGTAGAGCTACATGTGAACCCACGGCAGTTCAATTGCCCTGTA TGTGACTATGCAGCTTCCAAGAAGTGTAATCTACAGTATCACTTCAAATCTAAGCATCCTACTTGTCCTAATAAAA CAATGGATGTCTCAAAAGTGAAACTAAAGAAAACCAAAAAACGAGAGGCTGACTTGCCTGATAATATTACCAATGA AAAAACAGAAATAGAACAAACAAAAATAAAAGGGGATGTGGCTGGAAAGAAAAATGAAAAGTCCGTCAAAGCAGAG AAAAGAGATGTCTCAAAAGAGAAAAAGCCTTCTAATAATGTGTCAGTGATCCAGGTGACTACCAGAACTCGAAAAT CAGTAACAGAGGTGAAAGAGATGGATGTGCATACAGGAAGCAATTCAGAAAAATTCAGTAAAACTAAGAAAAGCAA AAGGAAGCTGGAAGTTGACAGCCATTCTTTACATGGTCCTGTGAATGATGAGGAATCTTCAACAAAAAAGAAAAAG AAGGTAGAAAGCAAATCCAAAAATAATAGTCAGGAAGTGCCAAAGGGTGACAGCAAAGTGGAGGAGAATAAAAAGC AAAATACTTGCATGAAAAAAAGTACAAAGAAGAAAACTCTGAAAAATAAATCAAGTAAGAAAAGCAGTAAGCCTCC TCAGAAGGAACCTGTTGAGAAGGGATCTGCTCAGATGGACCCTCCTCAGATGGGGCCTGCTCCCACAGAGGCGGTT CAGAAGGGGCCCGTTCAGGTGGAGCCGCCACCTCCCATGGAGCATGCTCAGATGGAGGGTGCCCAGATACGGCCTG CTCCTGACGAGCCTGTTCAGATGGAGGTGGTTCAGGAGGGGCCTGCTCAGAAGGAGCTGCTGCCTCCCGTGGAGCC TGCTCAGATGGTGGGTGCCCAAATTGTACTTGCTCACATGGAGCTGCCTCCTCCCATGGAGACTGCTCAGACGGAG GTTGCCCAAATGGGGCCTGCTCCCATGGAACCTGCTCAGATGGAGGTTGCCCAGGTAGAATCTGCTCCCATGCAGG TGGTCCAGAAGGAGCCTGTTCAGATGGAGCTGTCTCCTCCCATGGAGGTGGTCCAGAAGGAGCCTGTTCAGATAGA GCTGTCTCCTCCCATGGAGGTGGTCCAGAAGGAACCTGTTAAGATAGAGCTGTCTCCTCCCATAGAGGTGGTCCAG AAGGAGCCTGTTCAGATGGAGTTGTCTCCTCCCATGGGGGTGGTTCAGAAGGAGCCTGCTCAGAGGGAGCCACCTC CTCCCAGAGAGCCTCCCCTTCACATGGAGCCAATTTCCAAAAAGCCTCCTCTCCGAAAAGATAAAAAGGAAAAGTC TAACATGCAGAGTGAAAGGGCACGGAAGGAGCAAGTCCTTATTGAAGTTGGCTTAGTGCCTGTTAAAGATAGCTGG CTTCTAAAGGAAAGTGTAAGCACAGAGGATCTCTCACCACCATCACCACCACTGCCAAAGGAAAATTTAAGAGAAG AGGCATCAGGAGACCAAAAATTACTCAACACAGGTGAAGGAAATAAAGAAGCCCCTCTTCAGAAAGTAGGAGCAGA AGAGGCAGATGAGAGCCTACCTGGTCTTGCTGCTAATATCAACGAATCTACCCATATTTCATCCTCTGGACAAAAC TTGAATACGCCAGAGGGTGAAACTTTAAATGGTAAACATCAGACTGACAGTATAGTTTGTGAAATGAAAATGGACA CTGATCAGAACACAAGAGAGAATCTCACTGGTATAAATTCAACAGTTGAAGAACCAGTTTCACCAATGCTTCCCCC TTCAGCAGTAGAAGAACGTGAAGCAGTGTCCAAAACTGCACTGGCATCACCTCCTGCTACAATGGCAGCAAATGAG TCTCAGGAAATTGATGAAGATGAAGGCATCCACAGCCATGAAGGAAGTGACCTAAGTGACAACATGTCAGAGGGTA GTGATGATTCTGGATTGCATGGGGCTCGGCCAGTTCCACAAGAATCTAGCAGAAAAAATGCAAAGGAAGCCTTGGC AGTCAAAGCGGCTAAGGGAGATTTTGTTTGTATCTTCTGTGATCGTTCTTTCAGAAAGGGAAAAGATTACAGCAAA CACCTCAATCGCCATTTGGTTAATGTGTACTATCTTGAAGAAGCAGCTCAAGGGCAGGAGTAAAGCTGAATCTAAG TCGACTTAAGAACCGCTCGAGGCCGGCAAGGCCGGATCCAGACATGATAAGATACATTGATGAGTTTGGACAAACC ACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAA GCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTT TTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCTCATTCATCCTTTTCTCCTCGTCCCTCCT TCATTCATTCATAGCCCCCCGCCCTGCCCGCTTCAGCATTTCATTCATTCATTCATTCATTCATTTCCCGGAGCTC CGCTAGCGCACACCCCTTCAGCCGAAGGCCCCAGCGCGCAGGCGCAGGCCGGGAGAGGCAGGCACCCTCCAATCGT CGGGCGTCCTTCCTCCTCCGGGCGGCCGCCCGCTTCCCCATGAATGAACATTGACGTCAATGGGGCGGGGCGCGCC CACGTGACCCCGCGCGCTCCCCTTTATAAGGCGGTGGAGGCGCGGGCGCTGTCCAGCGTGCTGAAGCCGGAGCGAG CTAGCCGCCCGGAGCCGCGCCGACCCAGCTGAGCCCAGCCCACGGGACGCCAGACCTCGACCGTCGCTCCTACCCC GGCCACCGCTCGGAGCCGAGGCGGACGCGTCCCGATCTTCCCCTGTCCCCACCCTGCCCCGACCCTCCTCTCCACC TCTCGCGTCGTGACACCAGCTGGTAAATACTCCGCTGTTCGTCCCTCAAACCCTCGGCAGCCAGCCGTGGGCGTGA GGGAGGGTTCTCTCTCCTCTCGATGGGGGTGTTGCAAACACAGCGGGGAGCCCCCTGGTAAGGGTCCCCGGTAAAC GGGGGAGTCGCAGCTTTTTCTCTTGCTGCTGAAGTCGCCCACGCACCATCCGGGGAGTCCTACGGGGAGGGAGCAG AGATTTTTTTTTCCCCCATATTGCTGCTGCTTAGTACGTGGGCGATGGCAGTGAGATGGCTCAGGGAAGGGGCCGA GGAGGCCCTGGGTAAGCGAGGGCTTCGGGGGTTATTTTCCCATTTACACGGCTCCAGAGATCGGCACAACATCTTC CTCCTTTGCTCCTAAACGTTCCTCTTCTGGGTAAGGTTTGGGGGATCAGGGAAGCCCCGGGTTTCCTGCTGAAAGG TGGGGGAAGGGAACGTAGACCTAGAGAGGGGAATTCTTACAGAAATCCTCTTTTTTTGGTCCCTTCTATTTTTCAG TCTCCGGCAGCCTCTTGGTCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC CTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGC AGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA GGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTG GTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACT ACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAA CATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTG CCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGC TGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAACGCGTGAATTCACTCCTCA GGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTT CCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTT TCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACA TCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAA AGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGA TTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAG ATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCC AAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTG。

A kind of typical embodiment according to the present invention provides a kind of for detecting CRISPR/Cas9 gene editing system Cut the carrier that gene generates frameshift mutation.The carrier includes any of the above-described kind of Genetic elements.Genetic elements CIAs can connect Realize that stablizing for CIAs nucleic acid sequence is lost into pUC series or other cloned plasmids or other function property grain or host genome It passes, can be transferred in different hosts and be functioned with different carrier format passenger gene element CIAs.

A kind of typical embodiment according to the present invention, provides a kind of host cell.The host cell includes any of the above-described Kind generates the carrier of frameshift mutation for detecting CRISPR/Cas9 gene editing system cutting gene, such as host cell is big Enterobacteria.The carrier for being used to detect the cutting gene generation frameshift mutation of CRISPR/Cas9 gene editing system is transformed into large intestine In bacillus, plasmid is extracted from positive colony.Genetic elements CIAs can carry out the codon optimization of different plant species, can be not It functions with host cell.

A kind of typical embodiment according to the present invention provides a kind of detection CRISPR/Cas9 gene editing system cutting The method of gene generation frameshift mutation.This method includes using above-mentioned for detecting the cutting of CRISPR/Cas9 gene editing system The carrier that gene generates frameshift mutation is detected.

Preferably, comprising the following steps: above-mentioned carrier is inserted by cutting target dna sequence, then transfects to cell by S1 In system, while being transferred to the carrier for being mounted with gRNA；S2 observes the expression of the first reporter gene and the second reporter gene, root Judge whether gRNA produces shifting to the editor of target dna sequence according to the expression of the first reporter gene and the second reporter gene Code mutation.Preferably, when the second reporter gene normal expression, it is judged as that CRISPR/Cas9 gene editing system cuts gene Frameshift mutation is produced, and expands target dna sequence and is sequenced.Wherein, it is preferred that cell line is HEK293 cell line. HEK293 cell line is easy transfectional cell series, convenient for quickly detection.

Constructing Genetic elements CIAs of the invention facilitates gRNA/Cas9 complex cutting target DNA sequence in identification of cell The frameshift mutation situation generated after column and other possible pairing DNA sequence dnas, further to provide volume intracellular to the gRNA of design Volume information and provide background information for gene editing application.The pCIAs plasmid that the present invention constructs (includes said gene element Carrier, such as: SEQ ID NO:1) can be directly connected to target DNA sequence, transfect into HEK293 cell line, be transferred to simultaneously The carrier of the loading gRNA of design.After 48 hours, determined in fluorescin that fluorescence microscopy microscopic observation cell is expressed Whether gRNA produces frameshift mutation to the editor of target DNA sequence.After extracting DNA for the green fluorescence strain showed, PCR amplification target DNA sequence measures nucleic acid sequence after loading using carrier T, for identifying DNA sequence dna to be edited.

A kind of typical embodiment according to the present invention, Genetic elements CIAs is as shown in Figure 1, synthesized by DNA sequence dna Mode obtain.The CIAs sequence of synthesis needs to load CIAs to pUC19 plasmid by Sanger sequence verification.

A kind of typical embodiment according to the present invention, the application method of functional vector, comprising the following steps: A. is used PCR amplification linearizes CIAs；B. target fragment CIAs is connected in the pPuro plasmid of linearisation and obtains purpose plasmid CIAs-pPuro (or being pCIAs)；C. the step B plasmid obtained is converted into Escherichia coli, screens correct clone, And pass through Sanger sequence verification；D. after cutting pCIAs plasmid using Xba I and Kpn I digestion, connection target DNA sequence is extremely PCIAs plasmid.In one embodiment, it is transferred to different target DNA sequences respectively to pCIAs plasmid, and is transferred to gRNA carrier, comes It verifies cleavage activity and causes the base sequence after frameshift mutation.

The pCIAs plasmid can be used as the starting vector of testing goal DNA sequence dna, and the carrier have it is simple, quickly, The remarkable advantage of the genetic manipulations such as time saving, laborsaving, can be used for detecting gRNA to target DNA cleavage activity verifying and it is edited DNA sequence analysis confirmation.

The carrier that the present invention does the detection CRISPR/Cas9 gene editing system cutting gene generation frameshift mutation constructed can After whether being frameshift mutation and generate frameshift mutation with the mutation after the cutting of genome caused by the gRNA of quick detection design Generated base knocks out type and type probability.Method of the present invention can be quick, efficient, instant and stable detection The gRNA/Cas9 complex of design cuts postgenome, and whether the genotype repaired, which meets experiment, is expected, and grinds in gene function Studying carefully aspect has great significance.

In an exemplary embodiment of the invention, the technical advantage of the mechanism of action of Genetic elements CIAs is, by mesh DNA sequence dna load to pCIAs (pCIAs is the title that CIAs is connected on carrier) plasmid after, while turning gRNA carrier extremely In HEK293 cell line, it can quickly detect whether gRNA can cut target DNA sequence in time.Since pCIAs can be carried There is Puro resistance screening gene, so can remove the cell for not being transferred to purpose plasmid after addition puro, reduce high background Disadvantage.

In order to verify the cleavage activity and edited base sequence that Genetic elements CIAs quickly detects gRNA, the present invention In one embodiment, it loads in target DNA sequence to pCIAs, while turning gRNA vector plasmid and going to HEK293 cell together In system.After puro will be screened, the expression of fluorescence microscope reporter gene is used.If cell expression green is glimmering Light indicates that gRNA can cut target DNA sequence；If cell expresses red fluorescence, indicate that gRNA is cannot to cut mesh DNA sequence dna.In addition, target dna sequence base situation to be edited is identified by the DNA for extracting cell line, to analyze Edited result and determine suitable gRNA.

Beneficial effects of the present invention are further illustrated below in conjunction with embodiment.In the present invention for detailed description the step of or Reagent can be realized or be used the conventional reagent of this field using ordinary skill in the art means.

Embodiment 1

In the following embodiments, enzyme used is purchased from NEB, and HEK293 cell line matches the limited public affairs of shellfish biotechnology from Beijing Department.Genetic elements cmv enhancer, EF1 promoter, red fluorescent protein gene, REST gene, SV40pA terminator, S silencing The sequence information of son, green fluorescence protein gene and beta-globin polyadenylic acid comes from ncbi database, and synthesis is public by gold only intelligence Department completes, and (see Fig. 2 a) in reprinting to pUC19 plasmid.It is that Genetic elements CIAs illustrates map with reference to Fig. 1.

The present invention obtains pCIAs plasmid (Fig. 2 b) by genetic manipulation process, and example process is as follows:

The DNA of the plasmid pPuro of 300ng is mixed with 0.5 μ l restriction enzyme Xha I, carries out digestion, 37 DEG C of water-baths 1h obtains linearized vector segment pPuro after recycling.Using CIAs-pUC19 plasmid as template, with CIA-P1 (SEQ ID NO:2) It is primer amplification CIAs sequence with CIA-P2 (SEQ ID NO:3).Above-mentioned 2 segment is connected using SLIC cloning process.By 100ng PPuro and Insert Fragment CIAs according to molar ratio 1:2, handled at room temperature 2.5 minutes using the T4DNA polymerase of 0.5U, Subsequent ice bath is simultaneously converted into Escherichia coli, is coated on containing correct clone is screened on Amp plate, is obtained plasmid pCIAs, And carry out sequence verification.

SEQ ID NO:2

GTAAAACGACGGCCAGTGAATTCGACATTGATTATTGACTAGTTATTAATAG

SEQ ID NO:3

GCCAAGCTTGGGCTGCAGGTCGAGGGATCTCCATAAGAGAAG

Embodiment 2

Using H9 cell line genome DNA as template, primer target-p-1 (SEQ ID NO:4) and target- is used P-2 (SEQ ID N NO:5) carries out PCR reaction.Response procedures: 98 DEG C initial denaturation 5 minutes；98 DEG C of deformation 10s, 60 DEG C of annealing 15s, 72 DEG C of extension 1min, 35 circulations；72 DEG C of extension 10min.After recycling, target DNA sequence (as target sequence is obtained Column, SEQ ID NO:6).

By the DNA of the plasmid pCIAs of 300ng and restriction enzyme Xba I and Kpn I digestion (respectively 0.5 μ l and 0.5 μ l) mixing, digestion is carried out, 37 DEG C of water-bath 1h obtain linearized vector segment pCIAs after recycling.Use SLIC cloning process Connect above-mentioned 2 segment.By the pCIAs of 100ng and Insert Fragment target sequence according to molar ratio 1:2, the T4DNA of 0.5U is utilized Polymerase is handled 2.5 minutes at room temperature, and subsequent ice bath is simultaneously converted into Escherichia coli, is coated on containing screening on Amp plate Correct clone obtains plasmid target-pCIAs (Fig. 3 a), and carries out sequence verification.

In order to verify whether to be due to caused by DNA sequence dna as a result, being employed herein intragenic one section of HLA-E DNA sequence dna is as DNA sequence dna comparative experiments.According to the process described above, using primer target-1-p-1 (SEQ ID NO: 7) target-1 sequence (SEQ ID N NO:9) is obtained, load is connected to target-1-p-2 (SEQ ID N NO:8), amplification Plasmid target-1-pCIAs (Fig. 3 b) is obtained on body, and carries out sequence verification.

SEQ ID NO:4

GTCTAGACTCCCTGATCGCCTATAGATC

SEQ ID NO:5

CGGTACCCATGAAGAAAGCAGGTGTGGG

SEQ ID NO:6

CATGAAGAAAGCAGGTGTGGGTCCTGGACCAATAGCCCTCCTGAGGTCTGTCCTCAGGGACCTTCCCCT GTGACTTGTGACTGCTGGGATCAGGTCCCATCACCGCCGTAATCAAGGTGATAAATCTGTCCTTCATTTTAACAGGT GCTTTACAAAAGAGTAAGTGCTGGCACACAGGGCCCAGGCTGGGTCGGCCCATGATTGTGGAAGGTGCTTCCCAGTA ATGAGACAGGGCACATTTCTAGCTGGGGCTTGGAACCCTCAGTGAGACAAGAAATCTCAGACCCCACCCTTCACCCC TTCTCCACCTGAGCTCTTCCTCCTCCACATCACGGCAGCGACCACAGCTCCAGTGATCACAGCTCCAAGGAGAACCA GGCCAGCAATGATGCCCACGATGGGGATGGTGGGCTGGGAAGACAGCTCTGGGAAAAGAGGGGAAGGTGAGGGGCCC TGACCCTGCTAAAGGTCTCCAGAGAGGCTCCTGCTTTCCCTAAGAGACATGACACCCCCATCTCCCTCCTTACCCCA TCTCAGGGTGAGGGGCTTGGGCAGACCCTCATGCTGCACATGGCAGGTGTATCTCTGCTCCTCTCCAGAAGGCACCA CCACAGCCGCCCACTTCTGGAAGGTTCCATCCCCTGCAGGCCTGGTCTCCACGAGCTCCGTGTCCTGGGTCTGGTCC TCCCCATCCCGCTGCCAGGTCAGTGTGATCTCCGCAGGGTAGAAGCCCAGGGCCCAGCACCTCAGGGTGGCCTCATG GTCAGAGATGGGGTGGTGGGTCATATGTGTCTTGGGGGGGTCTGACGGGAAGAGTCAGAAAATTCAGGCATTTTGCA TCTGTCATGGGACACTCCACCAGCACGCATGTGGCCATCTTGAGAATGGACAGGACACCCGGGATGGGGAAGAGAGC ACAGAACCCAGACACCAGCCTGGACACAGGCACCTGGGATAATCTTCTATTCCCTGAGAAGGGAACAGCGACTTCTG GTCCTGACCTGAGTGGAGGCTGAGAGACTCAGAAGTGCTGGACTCAGACCCCCACACACATTGAGTGTGAAGCAGAG AACAAGGCCTGAGAGGAAAAGTCACGGGCCCAAGGCTGCTGCCGGTGTCAAAGGGAACCACTCATCAGTATTCGAGG GATCGTCTTCCCGTCACTCCTTCAGAGATTTTATCCCTTAATTGTGTCAGAGAGCAGGGCGGAACCTCAGAGTCACT CTCTGGTACAGGATCTGGAAACCCAGGAGGATTCCTCTCCCTCAGGACCAGAGGGAGGGTGATATTCTAGTGTTGGT CCCAATTGTCTCCCCTCCTTGTGGGAGGCCAGCCCGGGAGATCTATAGGCGATCAGGGAG

SEQ ID NO:7

CGGTACCGCAGACTCAGTTCTCATTCC

SEQ ID NO:8

GTCTAGAGCCTGTGTGGATGCTGAGTG

SEQ ID NO:9

GCAGACTCAGTTCTCATTCCCAATGGGTGTCGGGTTTCTAGAGAAGCCAATCAGCGTCGCCACGACTCC CGACTATAAAGTCCCCATCCGGACTCAAGAAGTTCTCAGGACTCAGAGGCTGGGATCATGGTAGATGGAACCCTCCT TTTACTCCTCTCGGAGGCCCTGGCCCTTACCCAGACCTGGGCGGGTGAGTGCGGGGTCGGGATGGAAACGGCCTCTA CCGGGAGTAGAGAGGGGCCGGCCCGGCGGGGGCGAAGGACTCGGGGAGCCGCGCCGGGAGGAGGGTCGGGCCGATCT CAGCCCCTCCTCGCCCCCAGGCTCCCACTCCTTGAAGTATTTCCACACTTCCGTGTCCCGGCCCGGCCGCGGGGAGC CCCGCTTCATCTCTGTGGGCTACGTGGACGACACCCAGTTCGTGCGCTTCGACAACGACGCCGCGAGTCCGAGGATG GTGCCGCGGGCGCCGTGGATGGAGCAGGAGGGGTCAGAGTATTGGGACCGGGAGACACGGAGCGCCAGGGACACCGC ACAGATTTTCCGAGTGAATCTGCGGACGCTGCGCGGCTACTACAATCAGAGCGAGGCCGGTGAGTGACCCCGGCCAG GGGAGCAGGTCACGACCCCTCCCCATCCCCCACGGACGGCGCGGGTCCCCTCGAATCTTCGGGTCCCAGATTCACCC CAAGGCTGCGGAACCCGCCCAGACCCTAGACCGGGGAGAGTCTCAGGCGCCTTTACCCGGTTCTTTTTCAGTTTAGG CCAAAATGCCCACAGGGTGGTGGCGACGGGGGCGGGGCTTGGTGGGCGGGACTGACTAAGGGGCGGGGCCAGGGTCT CACACCCTGCAGTGGATGCATGGCTGCGAGCTGGGGCCCGACGGGCGCTTCCTCCGCGGGTATGAACAGTTCGCCTA CGACGGCAAGGATTATCTCACCCTGAATGAGGACCTGCGCTCCTGGACCGCGGTGGACACGGCGGCTCAGATCTCCG AGCAAAAGTCAAATGATGCCTCTGAGGCGGAGCACCAGAGAGCCTACCTGGAAGACACATGCGTGGAGTGGCTCCAC AAATACCTGGAGAAGGGGAAGGAGACGCTGCTTCACCTGGGTAAGAGGGTCCACAGGGCTACTCTCCCATCTCCTTC TTGGGCTAGGACTGTGCCCACAGCTGACAGACCTCAAACAGTAGAAGAAACAGGGATGGAGGCCAGAATACCACTCC TCCCTTGGATCAGGAGAGGGAGCTGTCACCTGAGGTACAGGAGATCCTATACCACAGAGTGACTCTCTTAAAGGGCC AGACCTCTCTCAGGGGCAATTAAGGAATCTAGTCTCGCTGGAGATTCCATCCTTCAGATGAACTGATGAGCAGTTCT CTTTGACTCCCAGTATTAGGAATCACGGGGGAGTTTCTCTCGTGCCTGATTCTCAGCCCCACACCAAGAGTTTTTGG AGGTCTGACTCCAGCTTTTCTCAGTCACTCAGCATCCACACAGGC

Embodiment 3

For the cleavage activity of the gRNA of test-target gene.First choice needs to load target gRNA to carrier, required The operation wanted is as follows:

After carrying out gRNA design for target HLA-A gene in the present invention, primer gRNA12b-p-01 (SEQ ID is used NO:10 cycle of annealing) and after gRNA12b-p-02 (SEQ ID NO:11) mixing is executed in PCR instrument, obtains gRNA12b piece Section.

The DNA of the plasmid pHS-Cas 9 of 300ng is mixed with restriction enzyme Kpn I digestion (for 0.5 μ l), is carried out Digestion, 37 DEG C of water-bath 1h obtain linearized vector pHS-Cas 9 after recycling.

Above-mentioned product is connected using T4 ligase, and is converted into Escherichia coli, is coated on containing being screened on Amp plate Correct clone obtains plasmid gRNA12b-pHS-Cas 9 (Fig. 4 a), and carries out sequence verification.

When if necessary to detect more gRNA, then the carriers for being transferred to multiple loading gRNA is needed to can be realized.

In addition, be designed gRNAa to target gene HLA-A, the primer used be gRNAa-p-01 (SEQ ID NO: 12) and gRNAa-p-02 (SEQ ID NO:13), the site cut are to detect and work as purpose sequence not in target sequence It is that red fluorescent protein can be guided to express when arranging the not cleavage site of Cas9/gRNAa, used construction method is above-mentioned Construction method, the present invention construct plasmid gRNAa-pHS-Cas 9 (Fig. 4 b)

Meanwhile according to above design principle, to another section of DNA sequence (SEQ ID NO:9) gene editing in the present invention It is analyzed.Designed gRNA is respectively sg01RNA-pHS-Cas9 (Fig. 4 c) and sg04RNA-pHS-Cas9 (Fig. 4 d).

SEQ ID NO:10

GTGGAAAGGACGAAACACCGCGTGGAGACCAGGCCTGCAG

SEQ ID NO:11

GCTATTTCTAGCTCTAAAACCTGCAGGCCTGGTCTCCACG

SEQ ID NO:12

GTGGAAAGGACGAAACACCGTTACAAGTAAGACCTACTCC

SEQ ID NO:13

GCTATTTCTAGCTCTAAAACGGAGTAGGTCTTACTTGTAA

Embodiment 4

According to the experimental program of Lipofectamine 3000, by plasmid target-pCIAs, gRNA12b-pHS-Cas 9, target-1-pCIAs and sg01RNA-pHS-Cas9 is transferred in HEK293 cell line simultaneously respectively.37 degree of incubated cell 2-4 After it, in fluorescence microscopy microscopic observation cell, it is found that green fluorescence (Fig. 5 a and Fig. 5 c) is presented in cell, illustrate target dna sequence It is to produce frameshift mutation.

The DNA for extracting this cell line, using primer JD-p-1 (SEQ ID NO:14) and JD-p-2 (SEQ ID NO: 15) amplified fragments.Above-mentioned segment is connected using carrier T, converts into Escherichia coli, is coated on containing on Amp plate.By 20 After the random picking of clone spreads cultivation, sequencing company is sent to carry out sequencing verifying.

SEQ ID NO:14

GGTGCCCTCCATGTACAGC

SEQ ID NO:15

CTGCGCCGTTACAGATCCAA

Embodiment 5

According to embodiment 3 and method as described in example 4, plasmid gRNAa-pHS-Cas 9 (Fig. 4 b) and gRNAb- are constructed It pHS-Cas 9 (Fig. 4 d) and goes in HEK293 cell line with target-pCIAs, target-1-pCIAs simultaneously respectively.37 Degree is after incubated cell 2-4 days, in fluorescence microscopy microscopic observation cell, it is found that red fluorescence (Fig. 5 b and Fig. 5 d) is presented in cell, says Bright gRNA sequence does not cut target dna sequence.

It can be seen from the above description that the above embodiments of the present invention realized the following chievements:

1) compared to genome editor's situation in direct testing goal host, plasmid pCIAs constructed by the present invention, when Target DNA sequence is gone to after loading to pCIAs in HEK293 cell line, and the cleavage activity and analysis that can quickly detect gRNA are cut Base sequence after cutting；

2) being using advantage possessed by plasmid constructed by the present invention and detection method being capable of quick, easy detection purpose DNA sequence dna edits situation by different gRNA, can greatly save test period and operation link, reduces experimental cost, makes section Arriving for efficiency is ground to improve；

3) for more gRNA testing requirements, the method for the present invention is also that can provide detection gRNA cleavage activity and edited Genome editor's situation, and analysis OFF-target sequence can be carried out with the presence or absence of situation to be edited.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Sequence table

<110>Beijing Sai Bei Bioisystech Co., Ltd

<120>Genetic elements and the application of frameshift mutation are generated for detecting CRISPR/Cas9 gene editing system cutting gene

<130> PN90870SBSW

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 7703

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(7703)

<223>Genetic elements CIAs

<400> 1

tgtaaaacga cggccagtga attcgacatt gattattgac tagttattaa tagtaatcaa 60

ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 120

atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 180

ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 240

aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 300

tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 360

ctacttggca gtacatctac gtattagtca tcgctattac catggaatag caacagacat 420

acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttatcg atactagtaa 480

ggatctgcga tcgctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg 540

agaagttggg gggaggggtc ggcaattgaa cgggtgccta gagaaggtgg cgcggggtaa 600

actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt 660

atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac 720

agctgaagct tcgaggggct cgcatctctc cttcacgcgc ccgccgccct acctgaggcc 780

gccatccacg ccggttgagt cgcgttctgc cgcctcccgc ctgtggtgcc tcctgaactg 840

cgtccgccgt ctaggtaagt ttaaagctca ggtcgagacc gggcctttgt ccggcgctcc 900

cttggagcct acctagactc agccggctct ccacgctttg cctgaccctg cttgctcaac 960

tctacgtctt tgtttcgttt tctgttctgc gccgttacag atccaagctg tgaccggcgc 1020

ctacatgtct agannnnnnn nnnnnnnnnn nnnnnnnnnn nnnggtaccg tgtctaaggg 1080

cgaagagctg attaaggaga acatgcacat gaagctgtac atggagggca ccgtgaacaa 1140

ccaccacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca cccagaccat 1200

gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctaccag 1260

cttcatgtac ggcagcaaaa ccttcatcaa ccacacccag ggcatccccg acttctttaa 1320

gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag acgggggcgt 1380

gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat 1440

cagaggggtg aacttcccat ccaacggccc tgtgatgcag aagaaaacac tcggctggga 1500

ggcctccacc gagatgctgt accccgctga cggcggcctg gaaggcagaa gcgacatggc 1560

cctgaagctc gtgggcgggg gccacctgat ctgcaacttg aagaccacat acagatccaa 1620

gaaacccgct aagaacctca agatgcccgg cgtctactat gtggacagaa gactggaaag 1680

aatcaaggag gccgacaaag aaacctacgt cgagcagcac gaggtggctg tggccagata 1740

ctgcgacctc cctagcaaac tggggcacaa acttaattaa gccacccagg taatggggca 1800

gtcttctgga ggaggagggc tgtttaccag cagtggcaac attggaatgg ccctgcctaa 1860

cgacatgtat gacttgcatg acctttccaa agctgaactg gccgcacctc agcttattat 1920

gctggcaaat gtggccttaa ctggggaagt aaatggcagc tgctgtgatt acctggtcgg 1980

tgaagaaaga cagatggcag aactgatgcc ggttggggat aacaactttt cagatagtga 2040

agaaggagaa ggacttgaag agtctgctga tataaaaggt gaacctcatg gactggaaaa 2100

catggaactg agaagtttgg aactcagcgt cgtagaacct cagcctgtat ttgaggcatc 2160

aggtgctcca gatatttaca gttcaaataa agatcttccc cctgaaacac ctggagcgga 2220

ggacaaaggc aagagctcga agaccaaacc ctttcgctgt aagccatgcc aatatgaagc 2280

agaatctgaa gaacagtttg tgcatcacat cagagttcac agtgctaaga aattttttgt 2340

ggaagagagt gcagagaagc aggcaaaagc cagggaatct ggctcttcca ctgcagaaga 2400

gggagatttc tccaagggcc ccattcgctg tgaccgctgc ggctacaata ctaatcgata 2460

tgatcactat acagcacacc tgaaacacca caccagagct ggggataatg agcgagtcta 2520

caagtgtatc atttgcacat acacaacagt gagcgagtat cactggagga aacatttaag 2580

aaaccatttt ccaaggaaag tatacacatg tggaaaatgc aactattttt cagacagaaa 2640

aaacaattat gttcagcatg ttagaactca tacaggagaa cgcccatata aatgtgaact 2700

ttgtccttac tcaagttctc agaagactca tctaactaga catatgcgta ctcattcagg 2760

tgagaagcca tttaaatgtg atcagtgcag ttatgtggcc tctaatcaac atgaagtaac 2820

ccgccatgca agacaggttc acaatgggcc taaacctctt aattgcccac actgtgatta 2880

caaaacagca gatagaagca acttcaaaaa acatgtagag ctacatgtga acccacggca 2940

gttcaattgc cctgtatgtg actatgcagc ttccaagaag tgtaatctac agtatcactt 3000

caaatctaag catcctactt gtcctaataa aacaatggat gtctcaaaag tgaaactaaa 3060

gaaaaccaaa aaacgagagg ctgacttgcc tgataatatt accaatgaaa aaacagaaat 3120

agaacaaaca aaaataaaag gggatgtggc tggaaagaaa aatgaaaagt ccgtcaaagc 3180

agagaaaaga gatgtctcaa aagagaaaaa gccttctaat aatgtgtcag tgatccaggt 3240

gactaccaga actcgaaaat cagtaacaga ggtgaaagag atggatgtgc atacaggaag 3300

caattcagaa aaattcagta aaactaagaa aagcaaaagg aagctggaag ttgacagcca 3360

ttctttacat ggtcctgtga atgatgagga atcttcaaca aaaaagaaaa agaaggtaga 3420

aagcaaatcc aaaaataata gtcaggaagt gccaaagggt gacagcaaag tggaggagaa 3480

taaaaagcaa aatacttgca tgaaaaaaag tacaaagaag aaaactctga aaaataaatc 3540

aagtaagaaa agcagtaagc ctcctcagaa ggaacctgtt gagaagggat ctgctcagat 3600

ggaccctcct cagatggggc ctgctcccac agaggcggtt cagaaggggc ccgttcaggt 3660

ggagccgcca cctcccatgg agcatgctca gatggagggt gcccagatac ggcctgctcc 3720

tgacgagcct gttcagatgg aggtggttca ggaggggcct gctcagaagg agctgctgcc 3780

tcccgtggag cctgctcaga tggtgggtgc ccaaattgta cttgctcaca tggagctgcc 3840

tcctcccatg gagactgctc agacggaggt tgcccaaatg gggcctgctc ccatggaacc 3900

tgctcagatg gaggttgccc aggtagaatc tgctcccatg caggtggtcc agaaggagcc 3960

tgttcagatg gagctgtctc ctcccatgga ggtggtccag aaggagcctg ttcagataga 4020

gctgtctcct cccatggagg tggtccagaa ggaacctgtt aagatagagc tgtctcctcc 4080

catagaggtg gtccagaagg agcctgttca gatggagttg tctcctccca tgggggtggt 4140

tcagaaggag cctgctcaga gggagccacc tcctcccaga gagcctcccc ttcacatgga 4200

gccaatttcc aaaaagcctc ctctccgaaa agataaaaag gaaaagtcta acatgcagag 4260

tgaaagggca cggaaggagc aagtccttat tgaagttggc ttagtgcctg ttaaagatag 4320

ctggcttcta aaggaaagtg taagcacaga ggatctctca ccaccatcac caccactgcc 4380

aaaggaaaat ttaagagaag aggcatcagg agaccaaaaa ttactcaaca caggtgaagg 4440

aaataaagaa gcccctcttc agaaagtagg agcagaagag gcagatgaga gcctacctgg 4500

tcttgctgct aatatcaacg aatctaccca tatttcatcc tctggacaaa acttgaatac 4560

gccagagggt gaaactttaa atggtaaaca tcagactgac agtatagttt gtgaaatgaa 4620

aatggacact gatcagaaca caagagagaa tctcactggt ataaattcaa cagttgaaga 4680

accagtttca ccaatgcttc ccccttcagc agtagaagaa cgtgaagcag tgtccaaaac 4740

tgcactggca tcacctcctg ctacaatggc agcaaatgag tctcaggaaa ttgatgaaga 4800

tgaaggcatc cacagccatg aaggaagtga cctaagtgac aacatgtcag agggtagtga 4860

tgattctgga ttgcatgggg ctcggccagt tccacaagaa tctagcagaa aaaatgcaaa 4920

ggaagccttg gcagtcaaag cggctaaggg agattttgtt tgtatcttct gtgatcgttc 4980

tttcagaaag ggaaaagatt acagcaaaca cctcaatcgc catttggtta atgtgtacta 5040

tcttgaagaa gcagctcaag ggcaggagta aagctgaatc taagtcgact taagaaccgc 5100

tcgaggccgg caaggccgga tccagacatg ataagataca ttgatgagtt tggacaaacc 5160

acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 5220

tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 5280

tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 5340

ggtatggctg attatgatct cattcatcct tttctcctcg tccctccttc attcattcat 5400

agccccccgc cctgcccgct tcagcatttc attcattcat tcattcattc atttcccgga 5460

gctccgctag cgcacacccc ttcagccgaa ggccccagcg cgcaggcgca ggccgggaga 5520

ggcaggcacc ctccaatcgt cgggcgtcct tcctcctccg ggcggccgcc cgcttcccca 5580

tgaatgaaca ttgacgtcaa tggggcgggg cgcgcccacg tgaccccgcg cgctcccctt 5640

tataaggcgg tggaggcgcg ggcgctgtcc agcgtgctga agccggagcg agctagccgc 5700

ccggagccgc gccgacccag ctgagcccag cccacgggac gccagacctc gaccgtcgct 5760

cctaccccgg ccaccgctcg gagccgaggc ggacgcgtcc cgatcttccc ctgtccccac 5820

cctgccccga ccctcctctc cacctctcgc gtcgtgacac cagctggtaa atactccgct 5880

gttcgtccct caaaccctcg gcagccagcc gtgggcgtga gggagggttc tctctcctct 5940

cgatgggggt gttgcaaaca cagcggggag ccccctggta agggtccccg gtaaacgggg 6000

gagtcgcagc tttttctctt gctgctgaag tcgcccacgc accatccggg gagtcctacg 6060

gggagggagc agagattttt ttttccccca tattgctgct gcttagtacg tgggcgatgg 6120

cagtgagatg gctcagggaa ggggccgagg aggccctggg taagcgaggg cttcgggggt 6180

tattttccca tttacacggc tccagagatc ggcacaacat cttcctcctt tgctcctaaa 6240

cgttcctctt ctgggtaagg tttgggggat cagggaagcc ccgggtttcc tgctgaaagg 6300

tgggggaagg gaacgtagac ctagagaggg gaattcttac agaaatcctc tttttttggt 6360

cccttctatt tttcagtctc cggcagcctc ttggtcatgg tgagcaaggg cgaggagctg 6420

ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc 6480

agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc 6540

tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc 6600

gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc 6660

atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag 6720

acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc 6780

atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc 6840

cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc 6900

cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc 6960

atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg 7020

agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc 7080

gggatcactc tcggcatgga cgagctgtac aagtaaacgc gtgaattcac tcctcaggtg 7140

caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac 7200

tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg 7260

acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt 7320

ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg 7380

gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa 7440

agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc 7500

ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc 7560

ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca 7620

gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta 7680

atcatggtca tagctgtttc ctg 7703

<210> 2

<211> 52

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(52)

<223>primer CIA-P1

<400> 2

gtaaaacgac ggccagtgaa ttcgacattg attattgact agttattaat ag 52

<210> 3

<211> 42

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(42)

<223>primer CIA-P2

<400> 3

gccaagcttg ggctgcaggt cgagggatct ccataagaga ag 42

<210> 4

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(28)

<223>primer target-p-1

<400> 4

gtctagactc cctgatcgcc tatagatc 28

<210> 5

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(28)

<223>primer target-p-2

<400> 5

cggtacccat gaagaaagca ggtgtggg 28

<210> 6

<211> 1361

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1361)

<223>target DNA sequence

<400> 6

catgaagaaa gcaggtgtgg gtcctggacc aatagccctc ctgaggtctg tcctcaggga 60

ccttcccctg tgacttgtga ctgctgggat caggtcccat caccgccgta atcaaggtga 120

taaatctgtc cttcatttta acaggtgctt tacaaaagag taagtgctgg cacacagggc 180

ccaggctggg tcggcccatg attgtggaag gtgcttccca gtaatgagac agggcacatt 240

tctagctggg gcttggaacc ctcagtgaga caagaaatct cagaccccac ccttcacccc 300

ttctccacct gagctcttcc tcctccacat cacggcagcg accacagctc cagtgatcac 360

agctccaagg agaaccaggc cagcaatgat gcccacgatg gggatggtgg gctgggaaga 420

cagctctggg aaaagagggg aaggtgaggg gccctgaccc tgctaaaggt ctccagagag 480

gctcctgctt tccctaagag acatgacacc cccatctccc tccttacccc atctcagggt 540

gaggggcttg ggcagaccct catgctgcac atggcaggtg tatctctgct cctctccaga 600

aggcaccacc acagccgccc acttctggaa ggttccatcc cctgcaggcc tggtctccac 660

gagctccgtg tcctgggtct ggtcctcccc atcccgctgc caggtcagtg tgatctccgc 720

agggtagaag cccagggccc agcacctcag ggtggcctca tggtcagaga tggggtggtg 780

ggtcatatgt gtcttggggg ggtctgacgg gaagagtcag aaaattcagg cattttgcat 840

ctgtcatggg acactccacc agcacgcatg tggccatctt gagaatggac aggacacccg 900

ggatggggaa gagagcacag aacccagaca ccagcctgga cacaggcacc tgggataatc 960

ttctattccc tgagaaggga acagcgactt ctggtcctga cctgagtgga ggctgagaga 1020

ctcagaagtg ctggactcag acccccacac acattgagtg tgaagcagag aacaaggcct 1080

gagaggaaaa gtcacgggcc caaggctgct gccggtgtca aagggaacca ctcatcagta 1140

ttcgagggat cgtcttcccg tcactccttc agagatttta tcccttaatt gtgtcagaga 1200

gcagggcgga acctcagagt cactctctgg tacaggatct ggaaacccag gaggattcct 1260

ctccctcagg accagaggga gggtgatatt ctagtgttgg tcccaattgt ctcccctcct 1320

tgtgggaggc cagcccggga gatctatagg cgatcaggga g 1361

<210> 7

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(27)

<223>primer target-1-p-1

<400> 7

cggtaccgca gactcagttc tcattcc 27

<210> 8

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(27)

<223>primer target-1-p-2

<400> 8

gtctagagcc tgtgtggatg ctgagtg 27

<210> 9

<211> 1500

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(1500)

<223>target-1 sequence

<400> 9

gcagactcag ttctcattcc caatgggtgt cgggtttcta gagaagccaa tcagcgtcgc 60

cacgactccc gactataaag tccccatccg gactcaagaa gttctcagga ctcagaggct 120

gggatcatgg tagatggaac cctcctttta ctcctctcgg aggccctggc ccttacccag 180

acctgggcgg gtgagtgcgg ggtcgggatg gaaacggcct ctaccgggag tagagagggg 240

ccggcccggc gggggcgaag gactcgggga gccgcgccgg gaggagggtc gggccgatct 300

cagcccctcc tcgcccccag gctcccactc cttgaagtat ttccacactt ccgtgtcccg 360

gcccggccgc ggggagcccc gcttcatctc tgtgggctac gtggacgaca cccagttcgt 420

gcgcttcgac aacgacgccg cgagtccgag gatggtgccg cgggcgccgt ggatggagca 480

ggaggggtca gagtattggg accgggagac acggagcgcc agggacaccg cacagatttt 540

ccgagtgaat ctgcggacgc tgcgcggcta ctacaatcag agcgaggccg gtgagtgacc 600

ccggccaggg gagcaggtca cgacccctcc ccatccccca cggacggcgc gggtcccctc 660

gaatcttcgg gtcccagatt caccccaagg ctgcggaacc cgcccagacc ctagaccggg 720

gagagtctca ggcgccttta cccggttctt tttcagttta ggccaaaatg cccacagggt 780

ggtggcgacg ggggcggggc ttggtgggcg ggactgacta aggggcgggg ccagggtctc 840

acaccctgca gtggatgcat ggctgcgagc tggggcccga cgggcgcttc ctccgcgggt 900

atgaacagtt cgcctacgac ggcaaggatt atctcaccct gaatgaggac ctgcgctcct 960

ggaccgcggt ggacacggcg gctcagatct ccgagcaaaa gtcaaatgat gcctctgagg 1020

cggagcacca gagagcctac ctggaagaca catgcgtgga gtggctccac aaatacctgg 1080

agaaggggaa ggagacgctg cttcacctgg gtaagagggt ccacagggct actctcccat 1140

ctccttcttg ggctaggact gtgcccacag ctgacagacc tcaaacagta gaagaaacag 1200

ggatggaggc cagaatacca ctcctccctt ggatcaggag agggagctgt cacctgaggt 1260

acaggagatc ctataccaca gagtgactct cttaaagggc cagacctctc tcaggggcaa 1320

ttaaggaatc tagtctcgct ggagattcca tccttcagat gaactgatga gcagttctct 1380

ttgactccca gtattaggaa tcacggggga gtttctctcg tgcctgattc tcagccccac 1440

accaagagtt tttggaggtc tgactccagc ttttctcagt cactcagcat ccacacaggc 1500

<210> 10

<211> 40

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(40)

<223>primer gRNA12b-p-01

<400> 10

gtggaaagga cgaaacaccg cgtggagacc aggcctgcag 40

<210> 11

<211> 40

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(40)

<223>primer gRNA12b-p-02

<400> 11

gctatttcta gctctaaaac ctgcaggcct ggtctccacg 40

<210> 12

<211> 40

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(40)

<223>primer gRNAa-p-01

<400> 12

gtggaaagga cgaaacaccg ttacaagtaa gacctactcc 40

<210> 13

<211> 40

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(40)

<223>primer gRNAa-p-02

<400> 13

gctatttcta gctctaaaac ggagtaggtc ttacttgtaa 40

<210> 14

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(19)

<223>primer JD-p-1

<400> 14

ggtgccctcc atgtacagc 19

<210> 15

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<221> primer_bind

<222> (1)..(20)

<223>primer JD-p-2

<400> 15

ctgcgccgtt acagatccaa 20

Claims

1. a kind of Genetic elements for generating frameshift mutation for detecting CRISPR/Cas9 gene editing system cutting gene, special Sign is, the Genetic elements include the promoter effectively connected, the first reporter gene, REST gene, SV40pA gene, S heavy Silent son, the second reporter gene and terminator, wherein can be inserted between the promoter and the first reporter gene by cutting mesh DNA sequence dna is marked, first reporter gene is different from second reporter gene, and the expression of the REST gene is able to suppress institute State the expression of the second reporter gene behind S silencer.

2. Genetic elements according to claim 1, which is characterized in that the Genetic elements further include the enhancing effectively connected Son.

3. Genetic elements according to claim 2, which is characterized in that the enhancer is cmv enhancer.

4. Genetic elements according to claim 1, which is characterized in that first reporter gene is red fluorescent protein base Cause, second reporter gene are green fluorescence protein gene.

5. Genetic elements according to claim 1, which is characterized in that the promoter is EF1 promoter, the terminator For beta-globin polyadenylic acid.

6. Genetic elements according to claim 1, which is characterized in that the Genetic elements contain double enzyme site, described Double enzyme site be located at the promoter and it is described first report between, it is described can be connected to by cutting target dna sequence it is described Between double enzyme site.

7. Genetic elements according to claim 6, which is characterized in that the double enzyme site is Xba I and Kpn I digestion Site.

8. Genetic elements according to claim 1, which is characterized in that the REST gene and the S silencer amplification in Source of people H9 cell line.

9. Genetic elements according to claim 1, which is characterized in that the Genetic elements have such as SEQ ID NO:1 institute The nucleotide sequence shown.

10. a kind of carrier for generating frameshift mutation for detecting CRISPR/Cas9 gene editing system cutting gene, feature exist In the carrier includes Genetic elements as claimed in any one of claims 1-9 wherein.

11. carrier according to claim 10, which is characterized in that the carrier be pUC serial carrier, pEASY, PBlueScriptII or pBR322.

12. a kind of host cell, which is characterized in that including as described in claim 10 or 11 for detecting CRISPR/Cas9 Gene editing system cuts the carrier that gene generates frameshift mutation.

13. host cell according to claim 12, which is characterized in that the host cell is Escherichia coli.

14. a kind of method that detection CRISPR/Cas9 gene editing system cutting gene generates frameshift mutation, which is characterized in that Frameshift mutation is generated for detecting CRISPR/Cas9 gene editing system cutting gene using as described in claim 10 or 11 Carrier detected.

15. according to the method for claim 14, which comprises the following steps:

Carrier as described in claim 10 or 11 is inserted by cutting target dna sequence, then transfects into cell line by S1, It is transferred to the carrier for being mounted with gRNA simultaneously, the carrier for being mounted with gRNA also includes the DNA sequence dna for expressing Cas9 albumen；

S2 observes the expression of the first reporter gene and the second reporter gene, is reported according to first reporter gene and second The expression for accusing gene judges whether gRNA produces frameshift mutation to the editor of target dna sequence.

16. according to the method for claim 15, which is characterized in that further include: when the second reporter gene normal expression When, it is judged as that CRISPR/Cas9 gene editing system cutting gene produces frameshift mutation, and expand the target dna sequence It is sequenced.

17. according to the method for claim 15, which is characterized in that the cell line is HEK293 cell line.