CN113684212B - A method and application of MMEJ targeted genome modification based on palindromic sequence within the target site - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a target-site-internal-palindromic-sequence-mediated MMEJ-targeted genome modification method and application. The invention utilizes CRISPR/Cas9 system to carry out endogenous modification marking on fish genes by means of 6bp micro-homologous sequences formed after internal and external source identical targets are reversely complemented. Taking zebra fish as an example, the applicant carries out genome targeting modification on a gene myl specifically expressed by zebra fish hearts to establish a transgenic strain of the zebra fish heart specific marked green fluorescent protein. The MMEJ targeted genome modification based on the internal palindromic sequence of the target is an accurate and efficient repair mode, realizes tissue-specific marking on the premise of not damaging the functions of endogenous genes, truly, accurately and sensitively reflects the molecular expression level of the gene myl, and is further beneficial to promoting the research of the functions of the gene myl.

Description

Method for decorating MMEJ targeted genome based on target internal palindromic sequence mediation and application

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a target-site-internal-palindromic-sequence-mediated MMEJ-targeted genome modification method and application.

Background

The fine editing of genome is a key technology for realizing complex fine targeted modification of genome, which can insert artificially designed DNA fragments into genome at fixed points, wherein the DNA fragments can be single or multiple DNA molecules for encoding functional proteins, can be regulatory elements with transcriptional activity or genome sequences with regulatory activity, so that the targeted genome modification method is a key technology for realizing 'fixed point integration and controllable expression' of genes.

In recent years, with the rapid development of various targeted nuclease-mediated genome editing technologies in the field of biological genomes, CRISPR (clustered regularly interspersed short palindromic repeats)/Cas 9 system components are simpler and have higher efficiency, and become the most commonly used targeted nucleases at present. There have been researchers attempting to initially achieve site-directed integration in model animal zebra fish using targeted nuclease-induced host DNA double strand breaks (double strand breaks, DSBs), in combination with a variety of mechanisms of repair of DSBs. For example, the group Zhang Bo of Beijing university was first to achieve precise integration in zebra fish using targeted nuclease-mediated homologous recombination (Homologous Recombination, HR), but the efficiency of passage was only 1.5%, so that site-directed integration using HR was limited to limited genomic targets, and this technology has not been widely accepted and used in the field. Meanwhile, many research teams perform targeted integration by adjusting the length of homology arms or linearization of plasmids, etc., and although these studies report that gene knock-in is achieved at different targets, common problems in their studies have not been completely overcome, namely, passage efficiency and precise integration efficiency are low. In 2015, the China academy of sciences Du Jiulin skillfully designs the breaking position of the DSBs on the last intron of the target gene, and utilizes a non-homologous end joining (NHEJ) mechanism of the DSBs to realize successful introduction of exogenous genes without damaging endogenous gene expression, and the passage efficiency reaches 12 percent. Furthermore, they improved the efficiency of gene knock-in to 28% (2/7) in recent studies using this design concept. However, since introns often contain a large number of repeated sequences, it is disadvantageous to design a gRNA target, and random loss of DNA sequence by NHEJ often results in unsatisfactory integration and expression of the gene of interest. Therefore, the fish genome site-directed knock-in technology based on the HR mechanism and the NHEJ mechanism has various problems in aspects of target selection, vector construction, knock-in efficiency and the like.

In recent years, studies by the applicant and the haverso medical institute Alexander Schier teaching laboratories have successively found that micro-homology mediated terminal ligation (microhomology MEDIATED END joining, MMEJ) is the primary repair modality for DSBs during early development of fish. Then, by utilizing MMEJ mechanism to realize accurate integration in zebra fish embryo, under the condition that NHEJ mediated exogenous gene knocking-in efficiency is up to 56%, the efficiency of accurate integration of exogenous gene can be promoted to 60% -77% by introducing homologous fragments (10-40 bp) with different lengths near the target point. However, the methods are limited in the manner in which the vector DSBs are produced in the aforementioned reports, and the methods in these reports are limited in application range.

The living body imaging of fluorescent marker protein is an important tool for understanding gene functions, the most accurate method for reproducing the expression level and mode of physiological genes is to directly mark the genes at endogenous genome sites thereof to generate corresponding fusion proteins, and the key technology for realizing the specific marking is a targeted nuclease-mediated gene knock-in technology. Although gene knock-in technology is enabled in many model organisms using the CRISPR/Cas9 system, gene knock-in at multiple loci is also achieved in zebra fish, the reported methods still have certain limitations in terms of knock-in efficiency, accuracy and universality, and so far the strategies in these reports have not been applicable to all genomic targets. Therefore, there is a need to find a highly efficient and accurate universal gene knock-in technique.

Disclosure of Invention

The invention aims to provide a preparation method of a target genome modification based on a target internal palindromic sequence mediated MMEJ, which is specifically fixed-point gene knock-in.

The invention further aims at providing an application of the method for carrying out target genome modification based on the target internal palindromic sequence mediated MMEJ targeting in fish gene knock-in.

In order to achieve the above object, the present invention adopts the following technical measures:

a method for targeted genomic modification based on intra-target palindromic sequence mediated MMEJ, comprising the steps of:

1) Selecting target sequence containing palindromic sequence in target gene as gRNA, wherein the palindromic sequence is CCNMGG, NM represents AT, TA, CG or GC;

2) Amplifying a target knock-in gene, wherein the 5' end of the gene is provided with a reverse complementary sequence of the gRNA in the step 1), and constructing a gene knock-in vector carrying an exogenous gene after amplification;

3) The gRNA, gene knock-in vector, cas9 mRNA was microinjected into fish 1-cell stage embryos.

In the above steps, preferably, after the gRNA is selected, verification of the effectiveness of the target point is performed first, that is, the synthetic target point is injected into the zebra fish 1 cell stage by using the CRISPR/Cas9 system by means of microinjection, the injection components are Cas9 mRNA and gRNA, the genome is extracted after the embryo development after injection, and sequencing is performed to verify the effectiveness of the designed target point.

The method can be used for fish gene knock-in or fish gene endogenous markers.

In the above steps, preferably, CCGCGGCCAAGAGGGGGAAAACT of zebra fish myl gene is selected as gRNA, a gene knock-in vector pGT-myl7 (shown as SEQ ID NO. 1) is finally constructed, pGT-myl plasmid, cas9 mRNA, target gRNA and phenol red are mixed, and a sample is injected into zebra fish embryo of 1 cell stage by using a microinjection method, so that a target product can be obtained by screening.

In the above steps, preferably, the exogenous gene is an exogenous modification gene, preferably a fluorescent marker gene, including but not limited to a green fluorescent protein gene, a red fluorescent protein gene, a blue fluorescent protein gene, and the like.

Compared with the prior art, the invention has the following advantages and effects:

According to the invention, after the endogenous target is reversely complemented and is used as the target of an exogenous vector, after being cut by a CRISPR/Cas9 system, the internal and external source cutting tail ends can form 6bp micro-homologous sequences, the precise targeted modification of the endogenous gene can be realized by utilizing a MMEJ DSBs repairing mechanism by means of the micro-homologous sequences, and the exogenous vector sequence is successfully introduced to finish fluorescence or Tag marking of the endogenous gene. The design realizes that an endogenous high-efficiency target is an exogenous vector target, a 6bp micro-homology mediated DNA (deoxyribonucleic acid) connection mode has the accuracy of HR mediated gene typing and the high efficiency higher than NHEJ mediated gene typing, and the construction of MMEJ exogenous vectors is far more convenient and faster than that of HR exogenous vectors, and is not limited by the interference of single base polymorphism widely existing in endogenous genome like HR. Therefore, the method has obvious advantages in establishing the accurate and efficient gene knock-in technology in fish by utilizing MMEJ mediated DNA repair mechanism.

The exogenous vector is designed simply by utilizing the strategy, and very specific fluorescent protein expression can be observed in the generation P0, so that the strategy has higher knock-in efficiency.

Drawings

FIG. 1 is a schematic representation of a target at a genomic location;

Wherein, the designed target (CCGCGGCCAAGAGGGGGAAAACT, PAM) is positioned on myl gene exon 2, the palindromic sequence of the target is CCGCGG, and the target is verified to be an effective target through PCR product sequencing.

FIG. 2 is a schematic diagram of the constructed pGT-myl targeted modification of the exogenous vector;

The target (myl-TS-RC) sequence on the carrier is the reverse complementary sequence (AGTTTTCCCCCTCTTGGCCGCGG, PAM) of the endogenous target, and the targeting modified endogenous gene myl is complemented with the delta CDS on the exogenous carrier, so that the function of the endogenous gene is not destroyed.

FIG. 3 is a schematic representation of a MMEJ-targeted modification of the myl7 genome;

a is a strategy based on target internal palindromic sequence mediated MMEJ targeted genome modification, b is early embryo after P0 generation specific targeted modification, and specific green fluorescence expression can be seen in P0 generation. c is the early verification integration efficiency of the P0 generation embryo, d is the cloning analysis of the target band in the c graph, and the accurate integration efficiency can reach 80% (4/5) through sequencing.

FIG. 4 is a successfully screened F1 embryo;

a is F1 generation embryo of early myocardial cell specific expression fluorescent protein, b is specific imaging of myocardial cell, c is the efficiency (5/5) of accurate integration of F1 generation embryo.

Detailed Description

The technical scheme of the invention is conventional in the art unless specifically stated otherwise. All primers were synthesized by Beijing Optimuno technologies Co.

Example 1:

in the embodiment 1 of the invention, zebra fish myl gene (ENSDARG 00000019096) with palindromic sequence CCGCGG at exon is taken as an example for specific explanation, and other fish genes with palindromic sequence are also suitable for the method of the invention to achieve high-efficiency integration.

A target-based internal palindromic sequence mediated MMEJ-targeted genome modification method comprises the following steps:

(1) Verification of endogenous target with palindromic sequence features

A target (CCGCGGCCAAGAGGGGGAAAACT, PAM) with the sequence characteristics is found on the zebra fish myl gene exon 2 (FIG. 1). The effectiveness of the target spot is further verified by using a CRISPR/Cas9 system through in vitro transcription synthesis of the gRNA, wherein after Cas9 mRNA, the gRNA and phenol red are mixed, the mixed product is injected into embryos of a zebra fish 1 cell stage, and the injection dose is 500 ng/mu L of Cas9 mRNA and 100 ng/mu L of gRNA. Genome is extracted after embryo development after injection reaches 1 day, and the validity of the designed target point is detected by sending sequencing after PCR amplification by using myl-test-F and myl-test-R primers. Through sequencing, a set of peaks appear at the designed target position (figure 1), which shows that the target has higher knockout efficiency and is used as a target for subsequent gene knockout.

TABLE 1 primer sequences for myl Gene targeting modification in the present application

(2) Construction of Gene knock-in vector

According to the step (1), the sequence after reverse complementation of the endogenous effective target can be used as the target of the exogenous vector, so that the design is the micro-homologous end connection mediated by the palindromic sequence in the same target, and compared with the construction of the exogenous vector when the gene is knocked in by using the HR and NHEJ methods, the construction of the exogenous vector of MMEJ targeted modified genome can be realized by only adding the sequence after reverse complementation of the endogenous target (comprising the PAM region) to the primer of the amplified fragment. The effective target (CCGCGGCCAAGAGGGGGAAAACT, PAM) found on the myl gene exon 2 can be used as the target of an exogenous vector by adding the sequence (AGTTTTCCCCCTCTTGGCCGCGG, PAM) after reverse complementation into an upstream primer myl-FHR-F (AGTTTTCCCCCTCTTGGCCGCGGCGAAAAGAGGCAAGACCGCTCAAAGAGGCTCTTCCA) for amplifying myl DeltaCDS, amplifying fragments between myl gene targets and stop codons of the target fragment by using 3dpf zebra fish embryo cDNA as a template with myl-FHR-R primers, amplifying by using myl-VHR-F and myl-VHR-R primers and plasmid pMD18T-EGFP as a template in reverse PCR to obtain a vector skeleton, constructing the gene knock-in vector by using the homologous recombination method with the amplified vector skeleton to prepare a vector pGT-myl7, wherein the sequence of the vector pGT-myl7 is shown in SEQ ID NO.1 (figure 2), and the vector carries myl and EGFP genes simultaneously.

The plasmid pMD18T-EGFP is obtained by connecting a green fluorescent protein EGFP gene to pMD 18T.

(3) Microinjection, embryo early stage fluorescence screening and culture

The previously prepared pGT-myl plasmid, cas9 mRNA, target gRNA and phenol red were mixed and the samples were microinjected into 1-cell stage zebrafish embryos by microinjection using a microinjection apparatus, the final concentrations of each component being pGT-myl plasmid 50 ng/. Mu.L, cas9 mRNA 500 ng/. Mu.L and gRNA100 ng/. Mu.L (FIG. 3 a). Heart-specific expression of green fluorescent protein (b in fig. 3) was clearly observed at 2-3dpf and was highly efficient (16/50) after injection. The target band of the target modified genome can also be amplified by detecting the insertion event through the primer myl-test-F and EGFP-R2 (c in FIG. 3), the target band is purified and the linked T vector is recovered, and then the target band is subjected to single clone sequencing, and the sequencing result shows that the precision integration efficiency by MMEJ mode can reach 80% (4/5) (d in FIG. 3), and the integration efficiency by NHEJ is 20% (1/5). Therefore, the micro-homologous connection targeting genome modification mediated by the palindromic sequence in the same target point has higher accuracy and high efficiency.

However, it should be noted that, although the injected plasmid is extracted by the detoxification kit, more deaths or deformities still occur in the embryo after the plasmid-containing sample is injected, so that more embryos can be injected in early stage, and at least 50 healthy positive embryos can be selected later for passage establishment.

(4) Efficient germ line transfer of precise targeted genomic modifications

Carefully culturing the positive embryo in the step (3) to sexual maturity, and observing whether an embryo which specifically expresses green fluorescent protein by heart exists in the F1-generation embryo or not through lateral crossing with a wild adult fish so as to obtain a stably inherited gene knock-in strain. The P0 generation selects 5 positive fishes which specifically express green fluorescent protein through hearts, 1 strain fish which can be inherited stably is successfully screened, and the transfer efficiency of the germline is 22/150 (a in fig. 4), namely the number of positive embryos which appear from the P0 generation to the F1 generation is called the transfer efficiency of the germline, namely 22 positive embryos in 150F 1 generation embryos, and the specific green fluorescent protein is expressed in the central organs of early embryo (b in fig. 4). The 5-tail F1-generation positive embryo tail fin sampling is randomly selected in early embryo development period to detect the event of directional integration, and sequencing finds that all 5 tails are integrated in a predesigned way, and the accurate integration efficiency reaches 100% (5/5) (c in fig. 4). Further illustrates efficient germline transfer of targeted genomic modifications with precise targeted integration events mediated by palindromic sequences within the target.

Example 2:

MMEJ targeted genome modification method based on other palindromic sequence mediation in target point:

Using the procedure of example 1, the remaining palindromic sequence (CCNMGG, NM stands for AT, TA, CG) mediated MMEJ-targeted genomic modification procedure was validated and established in other genes of zebra fish. For example, the target with CCATGG sequence characteristic (CCTTCAAAGATCACCCTCCATGG) is found on the exon 10 of the mpx gene (ENSDARG 00000019521), the target with CCTAGG sequence characteristic (AGGTCTACAAAGACAGCCCTAGG) is found on the exon 2 of the tuba a gene (ENSDARG 00000001889), and the target with CCCGGG sequence characteristic (CTATGGACCGAAGCAGGCCCGGG) is found on the exon 6 of the cyp11a1 gene (ENSDARG 00000002347), and experiments prove that the targets with the sequence characteristic can well realize targeted modification on a target genome, so that the universality of the technology in application is further demonstrated. The primers used in the procedure for each gene are shown in Table 2, and specific strategies are described in example 1.

TABLE 2 primer sequences for other palindromic sequence targeting genome modification

Sequence listing

<110> Institute of aquatic organisms at national academy of sciences

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tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttgcatgc ctgcaggtcg 420

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ttccaatgtc ttctccatgt ttgagcaatc acaaatacag gagtttaagg aggcttttgg 540

ctgcatagat cagaaccggg atggagttat caacaaatct gatctgaagg aaacttatgc 600

acaactaggg aagctgaatg tgagtgatga agagctggag tccatgctaa cagaaggaaa 660

agggcccata aacttcactg tcttcctcac cctctttgga gagaagctca atggcacaga 720

cccagaggaa accatccttg ctgcttttaa attgttcgac cctaatgcca caggagttgt 780

caataaagat gagttcaaga ggctgctaat gacccaagct gataaattta cagcagaaga 840

ggttgaccag gcttttgcag tggctccaat agacgtggct ggaaatattg attataagtc 900

gctttgttac attatcacac atggtgatga aaaagaggaa tctggatccg gagccacgaa 960

cttctctctg ttaaagcaag caggagacgt ggaagaaaac cccggtcctg ccaccatggt 1020

gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga 1080

cgtaaacggc cacaagttca gcgtgtccgg cgagggagaa ggtgacgcta cgtatggcaa 1140

gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt 1200

gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca 1260

cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa 1320

ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa 1380

ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct 1440

ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat 1500

caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca 1560

ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct 1620

gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct 1680

ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaagaatt 1740

ctagatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag 1800

tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 1860

agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 1920

gaggtgtggg aggtttttta atctctagag gatccccggg taccgagctc gaattcgtaa 1980

tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 2040

cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 2100

attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 2160

tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 2220

ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 2280

gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 2340

ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 2400

cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 2460

ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 2520

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 2580

catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 2640

gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 2700

tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 2760

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 2820

actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 2880

gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 2940

aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 3000

gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 3060

aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 3120

atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca 3180

gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg 3240

atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca 3300

ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt 3360

cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt 3420

agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca 3480

cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca 3540

tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga 3600

agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact 3660

gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga 3720

gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg 3780

ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc 3840

tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga 3900

tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat 3960

gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt 4020

caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt 4080

atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac 4140

gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc 4200

tttcgtc 4207

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tgtaatacga ctcactataa gttttccccc tcttggccgg ttttagagct agaaatagc 59

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agcaccgact cggtgccact ttttc 25

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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tttgaggaaa ccgattgcta ca 22

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<213> Artificial sequence (ARTIFICIAL SEQUENCE)

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gtttgctcga atgccaatgt g 21

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ggatccggag ccacgaactt c 21

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agttttcccc ctcttggccg cggcgaaaag aggcaagacc gctcaaagag gctcttcca 59

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<212> DNA

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agttcacctt gatgccgttc tt 22

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tgtaatacga ctcactatac cttcaaagat caccctccag ttttagagct agaaatagc 59

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tgtaatacga ctcactataa ggtctacaaa gacagccctg ttttagagct agaaatagc 59

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tgtaatacga ctcactatac tatggaccga agcaggcccg ttttagagct agaaatagc 59

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cccacttgac tatccagccc 20

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cacggagcac aggatcaatg 20

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cacggagcac aggatcaatg 20

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cagctgacgg tatgtcccag tg 22

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gtgcaatact ctacaccgat c 21

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ccagtacacc aggatacttc c 21

Claims (7)

1. A method for targeted genomic modification based on intra-target palindromic sequence mediated MMEJ, comprising the steps of:

1) Selecting a target sequence containing a palindromic sequence in a target gene as gRNA based on the requirement of a CRISPR/Cas9 system on the sequence specificity of a motif region adjacent to a prosomain sequence, wherein the palindromic sequence is 6bp CCNMGG upstream of a Cas9 protein cleavage site, and NM represents AT, TA, CG or GC;

2) Amplifying a target knock-in gene, wherein the 5' end of the gene is provided with a reverse complementary sequence of the gRNA in the step 1), and constructing a gene knock-in vector carrying an exogenous gene after amplification;

3) The gRNA, gene knock-in vector, cas9 mRNA was microinjected into fish 1-cell stage embryos.

2. The method of claim 1, wherein the effectiveness of the target is verified after the gRNA is selected, namely, the synthesized target is injected into the cell stage of the zebra fish 1 by utilizing a CRISPR/Cas9 system by means of microinjection, the injected components are Cas9 mRNA and gRNA, the genome is extracted after the embryo development after the injection, and the effectiveness of the designed target is verified by sequencing.

3. The method of claim 1, wherein the exogenous gene is an exogenous modification gene.

4. The method of claim 3, wherein the exogenous modification gene is a fluorescent marker gene.

5. The method of claim 1, comprising the steps of selecting CCGCGGCCAAGAGGGGGAAAACT of zebra fish myl gene as gRNA, finally constructing a gene knock-in vector pGT-myl, wherein the gene knock-in vector is shown as SEQ ID NO.1, mixing pGT-myl7, cas9 mRNA, target gRNA and phenol red, injecting a sample into zebra fish embryo of 1 cell stage by using a microinjection method, and screening to obtain a target product.

6. Use of the method of claim 1 for fish gene knock-in.

7. Use of the method of claim 1 for the genetic modification of fish.