CN113061626B

CN113061626B - Method for knocking out zebra fish genes in tissue-specific manner and application

Info

Publication number: CN113061626B
Application number: CN201911295922.9A
Authority: CN
Inventors: 李佳; 杜久林; 李红羽
Original assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Current assignee: Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
Priority date: 2019-12-16
Filing date: 2019-12-16
Publication date: 2024-01-19
Anticipated expiration: 2039-12-16
Also published as: CN113061626A

Abstract

The invention provides a method for knocking out zebra fish genes in a tissue-specific manner and application thereof. In particular, the present invention provides a highly efficient and tissue-specific zebra fish gene knockout method and a nucleic acid construct for use in the method. The invention also provides a vector, a reagent and a host cell for expressing the nucleic acid construct. The invention can simply and conveniently obtain multiple transgenic effect, greatly improve transgenic efficiency, further simplify screening steps and simply and conveniently obtain the target transgenic zebra fish.

Description

Method for knocking out zebra fish genes in tissue-specific manner and application

Technical Field

The invention relates to the field of transgenic animals, in particular to a method for knocking out zebra fish genes in a tissue-specific manner and application thereof.

Background

Zebra fish are important model animals for studying development and disease. The juvenile fish is small, exquisite and transparent, and the tissue system has certain representativeness and conservation and certain complexity, so that the development and various physiological activities of the tissue organs can be observed dynamically under various operation means existing in a laboratory. To fully exploit the advantages of zebra fish in research, it is necessary to establish a tissue-specific gene knockout method for zebra fish. Currently, in zebra fish, methods for producing complete gene functional deletions are mature and have been widely used. However, techniques for specifically knocking out genes in specific tissues and cell types are still very immature, which is one of the bottlenecks currently restricting the development of the research field of zebra fish.

Thus, there is an urgent need in the art for a highly efficient and tissue-specific method of knockout of zebra fish genes to achieve knockout of gene function in specific tissues and cell types.

Disclosure of Invention

The invention aims to provide a zebra fish gene knockout method with high efficiency and tissue specificity, so as to realize gene knockout function in specific tissues and cell types.

In a first aspect of the invention, there is provided a nucleic acid construct I, said construct having the structure of formula I from 5 'to 3':

LA-X-RA (I)

in the method, in the process of the invention,

LA, X, RA are elements for constructing the construct, respectively;

each "-" is independently a bond or a nucleotide linking sequence;

LA is the left homologous arm sequence after modification;

x is a first exogenous gene expression cassette;

RA is a right homology arm sequence;

the element LA sequence and the RA sequence enable the construct to generate fixed-point non-homologous recombination with a target segment of a zebra fish chromosome, wherein the target segment is a zebra fish target gene 3 '-end containing an intron, an exon, a terminator and a 3' -UTR segment, and a single guide RNA (sgRNA) target sequence is contained in the intron sequence of the target segment;

the site of LA site-directed recombination is located in the 5' end of the target segment, from the introns and exons up to the pre-terminator sequence, and the LA sequence (5 '. Fwdarw.3 ') comprises the single guide RNA (sgRNA) target sequence and an operably linked nucleic acid construct II.

In another preferred embodiment, the sequence length of the target segment is 1000-6000bp.

In another preferred embodiment, the homology arm sequence is 200-5000bp, preferably 500-3000bp, more preferably 1000-2000bp in length.

In another preferred embodiment, the site of site-directed recombination of the target segment of nucleic acid construct I is located in the hey gene region on chromosome 20 of zebra fish, specifically at positions 39589569 to 39591030 of NC_007131.

In another preferred embodiment, the target segment of the nucleic acid construct I is exon 5E 5 of the zebra fish hey2 gene and an intron sequence comprising the sgRNA target sequence.

In another preferred embodiment, the sgRNA target sequence is 18-24nt in length; preferably 19-22nt.

In another preferred embodiment, the sgRNA target sequence targets an intron sequence of the hey gene.

In another preferred embodiment, the intron sequence, hey sgRNA target sequence, is GGAAGGATAATGGTTGGGT (SEQ ID NO: 2), wherein PAM is AGG.

In another preferred embodiment, the nucleic acid construct I has a nucleic acid sequence shown in SEQ ID NO. 1, wherein the nucleic acid sequence of the LA left homology arm sequence is 1-3309 of the nucleic acid sequence shown in SEQ ID NO. 1, the nucleic acid sequence of the first exogenous gene expression cassette X is 3310-4098 of the nucleic acid sequence shown in SEQ ID NO. 1, and the nucleic acid sequence of the RA right homology arm is 4099-5205 of the nucleic acid sequence shown in SEQ ID NO. 1. The sequence of SEQ ID NO. 1 is as follows:

atgatcttattttgactaagcgtggctatgaagcagaaaggaaggataatgagttgggtaggttaggtaagactttcttagacatgagtcaggtcaaagacaacagataattccataaaacatatgtattttttgtattctatagaattgtcattaatattcatgcaaaagatttcttaagtgacatttgcaaatcactccagttgtgtctttctttacattgttttccagttaaatttaggattgatttctgttatttatttagaataataattgtatattaataatgatattgggcagtatattgtactgtaccctgctgctggaagggtatccatatggggtggttcgcccagggtgttgttacacaagtgaaagagcatcgacagggtaatttgctagcttaccggtttacgcgtataacttcgtatagcatacattatacgaagttatccaagcttcaccatcgacccgaattgccaagcatcaccatcgacccataacttcgtatagtacacattatacgaagttatttcgaacgtaatacgactcactatagggcgaattggagctccaccggtggcggccgctctagaactagtggatcctggttctttccgcctcagaagccatagagcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatagaatgacacctactcagacaatgcgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacagatggctggcaactagaaggcacagtcgaggctgatcagcggtttctggttctttccgcctcagaagccatagagcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatagaatgacacctactcagacaatgcgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacagatggctggcaactagaaggcacagtcgaggctgatcagcgagctccaccgcggtcaattaagtttgtgccccagtttgctagggaggtcgcagtatctggccacagccacctcgtgctgctcgacgtaggtctctttgtcggcctccttgattctttccagtctgtggtccacatagtagacgccgggcatcttgaggttcttagcgggtttcttggatctgtatgtggtcttgaagttgcagatcaggtggcccccgcccacgagcttcagggccatgtcgcttctgccttccaggccgccgtcagcggggtacagcatctcggtgttggcctcccagccgagtgttttcttctgcatcacagggccgttggatgggaagttcacccctctgatcttgacgttgtagatgaggcagccgtcctggaggctggtgtcctgggtagcggtcagcacgcccccgtcttcgtatgtggtgactctctcccatgtgaagccctcagggaaggactgcttaaagaagtcggggatgccctgggtgtggttgatgaaggttctgctgccgtacatgaagctggtagccaggatgtcgaaggcgaaggggagagggccgccctcgaccaccttgattctcatggtctgggtgccctcgtagggcttgccttcgccctcggatgtgcacttgaagtggtggttgttcacggtgccctccatgtacagcttcatgtgcatgttctccttaatcagctcttcgcccttagacacgacgtcaggtccagggttctcctccacgtctccagcctgcttcagcaggctgaagttagtagctcttctcttcttccgaccgcgaagagtttgtcgatcgactgaaaaaaaaaagggaagagagagacacgtcagaaacacacacacactccggattagtgagatctgaataggaacttcataacttcgtataatgtatgctatacgaagttatccaagcatcaccatcgaccctctagtccagaactcaccatcgacccataacttcgtataatgtgtactatacgaagttatactagtattatgtacctgactgatcgatttgcctttgatttctggcatttgtcgggaatttctcaaaacctgttgtcgagtcaaaatctgggctaaaatcatacagtctgaactcggctttaggggttaataatattgaccttaaaatggttttaaaagaattaaaaactgcttttattctagctgacataaaacaaataagactttctccagaagaaaaaaatattttaggaattacagtaaaaaatgtcttgctctgttaaacatcatttgggaaatatttgaacaaaggtatcaaaattcacaggaggtgtgtgtatttaaagattcactagtatgctcatttgaataattctcaatattttttgtcaggatatttcgacgctcattctctggccatggacttcttgagcatcggcttccgggagtgtctgactgaagtggccaggtatttgagctctgtggaaggcctggactccagcgaccctctccgtgtccgtctggtttctcacctcagcagctgtgcctcgcagagggaagcagccgccatgaccacatccatagcccatcaccagcaggcccttcacccgcaccactgggctgccgctttgcatcccattcctgctgcgttcctgcagcagagcggacttccctcctcagagagctcctccggcaggctgtctgaggctcctcaaagaggtgcagcccttttctcccatagtgactcggcactcagagcgccctctactggaagtgtggctccttgcgtgccaccgctgtccacttctctgctttcgttatcagcgaccgttcatgcagcagctgctgcagctgcagctcaaaccttccctctatcatttcccgctggattcccactcttcagccccagcgttacagcatcttcagtggcttcttccaccgtgagctcttccgtttccacatccaccacatcccaacagagcagcgggagcaacagtaaaccataccgaccgtggggaactgaagtgggagcgttttcgggaggtggatccggagctactaatttctccttgcttaagcaagctggtgatgttgaagaaaatcctggtcctatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaccggttaaatgttggatttaaatgttggacgtcttccatgctttgtacataaaggaaagcagcggctattgtgcctgcttcggtcagcagcatgggcttttgtcttcctctacacttgtgcacatatgcagcgtcaaacttaagccaacattctgggaagaaaagaaagagtttttacacgtcgcactgtgttggaaaccgtaaaggaagtttgtttctgttttaacagtgcctgcataaacactgctaacatgctgcatttgagatgtatgctttgatatcatctgacttccacaaacacccaacagcagctttagagtgaacagcttgttctgaaacaaaccaaagttttgcagataatcactaaagtgaggtgtttgtttttttatctctgatttaacaatccagtttgtaaatctgtacatgtgtaagattgtaactagagtttatattgaaattagttcattggtatgatgcacttcaatcactactgtttgtttggggggagacaggatcttctccgatttatacaataggcctactgaagttgtttttttaaaataacattcactaatactcatgtgagatttttctactactgtaactgtgttaataaccaccctctgtaagatgtaaccttttcctatgcaaaaaaacaaatgtccctcaagaacgaactgagtgtgttttgttttcattctgacacacgctaataaaaccatccttccactagccttcaccacaacacatcgtggaatgttatgagagaaagtaattgttttcccaaagcattatttgagttcttgaaatcgtatggtagggaacaaatgtttgtgctctttaatgtgtttttctaataatgcaaaatatgcagatgaagtcaaacaaacagctgcaattgtaaccgccacttcaacagttataaatctgtcgacaaactttaaagaaagctacaaacacatttaatgaataaaaggtcatcattcttacatgatcagcagcaaatcggtttactttcattgaaaaaagtcaataatttcttctaaagctaaaataactttttagctgtgtgtgaagagctgtactgtgtgacggtgcctgctaaaacccta。

In another preferred embodiment, the first exogenous gene expression cassette X contains a GSG-P2A self-cleaving sequence and an EGFP sequence.

In another preferred embodiment, the EGFP sequence is as follows:

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag(SEQ ID NO.:3)。

in another preferred embodiment, the RA right homology arm sequence starts at the stop codon of the target gene and covers the 3 'gene spacer of the entire 3' UTR sequence of the target gene.

In another preferred embodiment, said LA left homology arm sequence comprises said hey sgRNA target sequence and a stretch of operably linked nucleic acid construct II.

In another preferred embodiment, the operably linked nucleic acid construct II has a structure of formula II from 5 'to 3':

L5-L5'-Y-L3-L3' (II)

in the method, in the process of the invention,

l5, L5', X, L, L3' are elements for constructing the construct, respectively;

each "-" is independently a bond or a nucleotide linking sequence;

l5 is a 5' first site-specific recombination sequence;

l5 'is a 5' second site-specific recombination sequence;

y is an inverted second exogenous gene expression cassette;

l3 is a 3' first site-specific recombination sequence;

l3 'is a 3' second site-specific recombination sequence;

and the site of RA site-directed recombination is located at the terminator and 3'UTR sequence of the 3' end of the target segment.

In another preferred embodiment, the nucleic acid sequence of the nucleic acid construct II is set forth in SEQ ID NO. 1 at positions 468-2271.

In another preferred embodiment, the inverted second exogenous gene expression cassette Y sequence comprises a BGHpA signal sequence, an inverted TagRFP sequence, and a cleavage acceptor; preferably, the sequence is positions 1280-2117 of the nucleic acid sequence set forth in SEQ ID NO. 1.

In another preferred embodiment, the BGHpA signal sequence is as follows:

ccatagagcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaata gaatgacacctactcagacaatgcgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcac gggggaggggcaaacaacagatggctggcaactagaaggcacag(SEQ ID NO.:7)。

in another preferred embodiment, the amino acid sequence of the TagRFP sequence is as follows:

VSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKVVEGGPLPFAFDILATSFMYGSRTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEANTEMLYPADGGLEGRSDMALKLVGGGHLICNFKTTYRSKKPAKNLKMPGVYYVDHRLERIKEADKETYVEQHEVAVARYCDLPSKLGHKLN(SEQ ID NO.:8)。

in another preferred embodiment, the site-specific recombination sequence is selected from the group consisting of: wild-type Loxp, and mutant Loxp.

In another preferred embodiment, the first site-specific recombination sequence is a wild-type loxP site.

In another preferred embodiment, the second site-specific recombination sequence is a mutant lox5171 site.

In another preferred embodiment, the nucleic acid construct I has the structure of formula III from 5 'to 3':

Z0-Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10-Z11 (III)

in the method, in the process of the invention,

z0 is an additional sequence (e.g., a vector-derived sequence, a selective endonuclease site sequence, or a combination thereof, etc.) that is absent or located at the 5' end;

z1 is a first left homology arm sequence comprising a single guide RNA (sgRNA) target sequence;

Z2 is a 5' first site-specific recombination sequence (equal to or identical to L5);

z3 is a 5 'second site-specific recombination sequence (equal to or identical to L5');

z4 is an inverted second exogenous gene expression cassette (equal or identical to Y);

z5 is a 3' first site-specific recombination sequence (equal to or identical to L3);

z6 is a 3 'second site-specific recombination sequence (equal to or identical to L3');

z7 is a second left homology arm sequence, wherein the first left homology arm sequence and the second left homology arm sequence are adjacent or contiguous on the genome to be genomically edited, and the first left homology arm sequence and the second left homology arm sequence together comprise a left homology arm, the left homology arm corresponding to the left arm (region) of the genome;

z8 is a coding sequence for encoding a P2A peptide or a functional peptide analogue thereof (e.g., GSG-P2A);

z9 is a first exogenous gene expression cassette;

z10 is a right homology arm sequence, which corresponds to the right arm (region) of the genome; (preferably, the boundaries of the left and right homology arms are (a) the end of the last exon of the gene to be knocked out; or a nonfunctional genomic sequence (e.g., 3' -URT, etc.));

z11 is an additional sequence (e.g., a vector-derived sequence, a selective endonuclease site sequence, or a combination thereof, etc.) that is absent or located at the 3' end;

Each "-" is independently a bond or a nucleotide linking sequence.

In another preferred embodiment, the first exogenous gene is a gene encoding a fluorescent protein.

In another preferred embodiment, the second exogenous gene is a gene encoding a fluorescent protein.

In another preferred embodiment, the wild-type loxP site is as follows:

ataacttcgtataatgtatgctatacgaagttat(SEQ ID NO.:4)。

in another preferred embodiment, the mutant lox5171 sequence is as follows:

ataacttcgtataatgtgtactatacgaagttat(SEQ ID NO.:5)。

in another preferred embodiment, the exogenous gene expression cassette includes a polynucleotide sequence of the exogenous gene and a sequence module for expressing the desired element.

In another preferred embodiment, the exogenous gene expression cassette further comprises a protein cleavage sequence.

In another preferred embodiment, the protein cleavage sequence is selected from the group consisting of: P2A sequences, and/or GSG-P2A sequences.

In another preferred embodiment, the nucleic acid sequence of the P2A sequence is as follows:

aggtccagggttctcctccacgtctccagcctgcttcagcaggctgaagttagtagc(SEQ ID NO.:6)。

in another preferred embodiment, the nucleic acid sequence of GSG-P2A encodes a glycine (glycine) -serine (serine) -glycine (glycine) -P2A peptide.

In another preferred embodiment, the exogenous gene is selected from the group consisting of: a reporter gene, a structural gene, a functional gene, or a combination thereof.

In another preferred embodiment, the reporter gene is selected from the group consisting of: fluorescent proteins, and variants or derivatives thereof (e.g., optically highlighting fluorescent proteins).

In another preferred embodiment, the fluorescent protein is selected from the group consisting of: green Fluorescent Protein (GFP), yellow Fluorescent Protein (YFP), red Fluorescent Protein (RFP) (e.g., mCherry), blue Fluorescent Protein (BFP), cyan fluorescent protein gene (CFP), orange fluorescent protein, photo-activated protein (FHP), photo-activated green cherry (GPAC), tomato red protein, photoprotein (e.g., aequorin, firefly luciferin), or combinations thereof.

In another preferred embodiment, the construct is linear or non-linear (e.g., circular).

In a second aspect of the invention there is provided a vector comprising a construct according to the first aspect.

In another preferred embodiment, the carrier is hey2 ^zCKOIS A donor plasmid having the sequence set forth in SEQ ID No.: 9:

tgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcggtacccactcgtcgacaaaactagggtttatatgatgatgatcttattttgactaagcgtggctatgaagcagaaaggaaggataatgagttgggtaggttaggtaagactttcttagacatgagtcaggtcaaagacaacagataattccataaaacatatgtattttttgtattctatagaattgtcattaatattcatgcaaaagatttcttaagtgacatttgcaaatcactccagttgtgtctttctttacattgttttccagttaaatttaggattgatttctgttatttatttagaataataattgtatattaataatgatattgggcagtatattgtactgtaccctgctgctggaagggtatccatatggggtggttcgcccagggtgccatttaaactagaaccatcactgcttcagatggcgtctttaatagttcacattgtatgattgttacacaagtgaaagagcatcgacagggtaatttgctagcttaccggtttacgcgtataacttcgtatagcatacattatacgaagttatccaagcttcaccatcgacccgaattgccaagcatcaccatcgacccataacttcgtatagtacacattatacgaagttatttcgaacgtaatacgactcactatagggcgaattggagctccaccggtggcggccgctctagaactagtggatcctggttctttccgcctcagaagccatagagcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatagaatgacacctactcagacaatgcgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacagatggctggcaactagaaggcacagtcgaggctgatcagcggtttctggttctttccgcctcagaagccatagagcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatagaatgacacctactcagacaatgcgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacagatggctggcaactagaaggcacagtcgaggctgatcagcgagctccaccgcggtcaattaagtttgtgccccagtttgctagggaggtcgcagtatctggccacagccacctcgtgctgctcgacgtaggtctctttgtcggcctccttgattctttccagtctgtggtccacatagtagacgccgggcatcttgaggttcttagcgggtttcttggatctgtatgtggtcttgaagttgcagatcaggtggcccccgcccacgagcttcagggccatgtcgcttctgccttccaggccgccgtcagcggggtacagcatctcggtgttggcctcccagccgagtgttttcttctgcatcacagggccgttggatgggaagttcacccctctgatcttgacgttgtagatgaggcagccgtcctggaggctggtgtcctgggtagcggtcagcacgcccccgtcttcgtatgtggtgactctctcccatgtgaagccctcagggaaggactgcttaaagaagtcggggatgccctgggtgtggttgatgaaggttctgctgccgtacatgaagctggtagccaggatgtcgaaggcgaaggggagagggccgccctcgaccaccttgattctcatggtctgggtgccctcgtagggcttgccttcgccctcggatgtgcacttgaagtggtggttgttcacggtgccctccatgtacagcttcatgtgcatgttctccttaatcagctcttcgcccttagacacgacgtcaggtccagggttctcctccacgtctccagcctgcttcagcaggctgaagttagtagctcttctcttcttccgaccgcgaagagtttgtcgatcgactgaaaaaaaaaagggaagagagagacacgtcagaaacacacacacactccggattagtgagatctgaataggaacttcataacttcgtataatgtatgctatacgaagttatccaagcatcaccatcgaccctctagtccagaactcaccatcgacccataacttcgtataatgtgtactatacgaagttatactagtattatgtacctgactgatcgatttgcctttgatttctggcatttgtcgggaatttctcaaaacctgttgtcgagtcaaaatctgggctaaaatcatacagtctgaactcggctttaggggttaataatattgaccttaaaatggttttaaaagaattaaaaactgcttttattctagctgacataaaacaaataagactttctccagaagaaaaaaatattttaggaattacagtaaaaaatgtcttgctctgttaaacatcatttgggaaatatttgaacaaaggtatcaaaattcacaggaggtgtgtgtatttaaagattcactagtatgctcatttgaataattctcaatattttttgtcaggatatttcgacgctcattctctggccatggacttcttgagcatcggcttccgggagtgtctgactgaagtggccaggtatttgagctctgtggaaggcctggactccagcgaccctctccgtgtccgtctggtttctcacctcagcagctgtgcctcgcagagggaagcagccgccatgaccacatccatagcccatcaccagcaggcccttcacccgcaccactgggctgccgctttgcatcccattcctgctgcgttcctgcagcagagcggacttccctcctcagagagctcctccggcaggctgtctgaggctcctcaaagaggtgcagcccttttctcccatagtgactcggcactcagagcgccctctactggaagtgtggctccttgcgtgccaccgctgtccacttctctgctttcgttatcagcgaccgttcatgcagcagctgctgcagctgcagctcaaaccttccctctatcatttcccgctggattcccactcttcagccccagcgttacagcatcttcagtggcttcttccaccgtgagctcttccgtttccacatccaccacatcccaacagagcagcgggagcaacagtaaaccataccgaccgtggggaactgaagtgggagcgttttcgggaggtggatccggagctactaatttctccttgcttaagcaagctggtgatgttgaagaaaatcctggtcctatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagaccggttaaatgttggatttaaatgttggacgtcttccatgctttgtacataaaggaaagcagcggctattgtgcctgcttcggtcagcagcatgggcttttgtcttcctctacacttgtgcacatatgcagcgtcaaacttaagccaacattctgggaagaaaagaaagagtttttacacgtcgcactgtgttggaaaccgtaaaggaagtttgtttctgttttaacagtgcctgcataaacactgctaacatgctgcatttgagatgtatgctttgatatcatctgacttccacaaacacccaacagcagctttagagtgaacagcttgttctgaaacaaaccaaagttttgcagataatcactaaagtgaggtgtttgtttttttatctctgatttaacaatccagtttgtaaatctgtacatgtgtaagattgtaactagagtttatattgaaattagttcattggtatgatgcacttcaatcactactgtttgtttggggggagacaggatcttctccgatttatacaataggcctactgaagttgtttttttaaaataacattcactaatactcatgtgagatttttctactactgtaactgtgttaataaccaccctctgtaagatgtaaccttttcctatgcaaaaaaacaaatgtccctcaagaacgaactgagtgtgttttgttttcattctgacacacgctaataaaaccatccttccactagccttcaccacaacacatcgtggaatgttatgagagaaagtaattgttttcccaaagcattatttgagttcttgaaatcgtatggtagggaacaaatgtttgtgctctttaatgtgtttttctaataatgcaaaatatgcagatgaagtcaaacaaacagctgcaattgtaaccgccacttcaacagttataaatctgtcgacaaactttaaagaaagctacaaacacatttaatgaataaaaggtcatcattcttacatgatcagcagcaaatcggtttactttcattgaaaaaagtcaataatttcttctaaagctaaaataactttttagctgtgtgtgaagagctgtactgtgtgacggtgcctgctaaaaccctactgcaggcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtc(SEQ ID NO.:9)。

in a third aspect of the invention, there is provided an agent, characterized in that the agent comprises (a) Cas9 mRNA, (b) target gene sgRNA, and (c) the nucleic acid construct of the first aspect and/or the vector of the second aspect, wherein the single guide RNA target sequence contained in the nucleic acid construct and/or the vector corresponds to (b) target gene sgRNA sequence.

In another preferred embodiment, the Cas9 mRNA sequence is optimized according to zebra fish codon preference.

In another preferred embodiment, the Cas9 mRNA sequence is zCas9 mRNA, the nucleic acid sequence of which is set forth in SEQ ID No.: 10:

ggatcacgacatcgactacaaagacgacgatgataagatggcccctaagaaaaagagaaaggtcggaattcacggagttcccgctgcagataaaaagtacagcattggactggacatcggaacaaatagcgtgggctgggctgtgattactgacgaatataaggtgcctagcaaaaagtttaaagtgctgggaaacaccgacagacacagcatcaaaaaaaacctgatcggcgctctgctgtttgatagcggtgaaactgccgaggctactagactgaagagaactgctagaagaagatataccagaagaaagaatagaatttgttacctgcaagaaatctttagcaatgagatggcaaaggttgacgatagcttctttcatagactggaggagagcttcctggtcgaggaggacaagaagcacgagagacaccccatcttcggaaatatcgtggacgaggtggcataccatgaaaagtatcctaccatttaccacctgagaaaaaagctggtggacagcacagacaaggccgatctgagactgatctacctggcactggcccacatgatcaaatttagaggccatttcctgattgaaggagacctgaaccccgataacagcgatgttgataaactgttcatccaactggttcagacctataaccaactgtttgaggagaaccctattaacgccagcggagtggatgcaaaggccatcctgagcgctagactgagcaaaagcagaagactggaaaatctgatcgcccagctgcccggcgaaaaaaagaatggactgttcggcaatctgattgcactgagcctgggactgacacctaacttcaagagcaatttcgatctggctgaggacgccaaactgcagctgagcaaagacacatatgatgacgacctggataacctgctggcacaaattggtgaccaatacgctgacctgttcctggctgctaagaatctgagcgatgccattctgctgagcgacatcctgagagtgaacacagagattaccaaggcacccctgagcgcaagcatgattaagagatacgacgagcaccaccaagatctgaccctgctgaaggccctggtcagacaacaactgccagagaagtataaagaaattttctttgaccaaagcaagaacggttacgctggctacattgacggcggtgcaagccaagaggagttctataagttcattaagccaatcctggagaaaatggatggaactgaggagctgctggttaagctgaatagagaggatctgctgagaaaacaaagaacattcgacaacggtagcatcccacaccagattcatctgggtgagctgcacgcaattctgagaagacaggaagacttttatccattcctgaaggacaacagagaaaagatcgagaagattctgacatttagaatcccctactacgtgggacctctggctagaggcaatagcagattcgcatggatgactagaaagagcgaggagacaattaccccttggaactttgaagaagtggtggataagggagcaagcgcccaaagcttcattgagagaatgacaaacttcgataagaacctgcctaacgagaaggttctgcccaagcatagcctgctgtatgaatatttcacagtgtacaacgagctgacaaaggtcaagtacgtcacagagggcatgagaaagcccgcctttctgagcggagaacaaaagaaggctattgttgacctgctgttcaagaccaacagaaaagttacagttaaacagctgaaagaggactacttcaaaaagattgaatgttttgacagcgtggaaatcagcggcgttgaggacagatttaacgctagcctgggcacctaccacgatctgctgaaaatcatcaaagataaggactttctggacaacgaagaaaacgaggacattctggaagacattgtgctgacactgactctgttcgaagatagagaaatgatcgaggaaagactgaaaacttatgcacatctgttcgacgacaaagtgatgaagcaactgaagagaagaagatacactggatggggcagactgagcagaaagctgatcaacggaatcagagacaagcaaagcggaaaaactattctggattttctgaaaagcgacggtttcgccaatagaaacttcatgcaactgattcacgatgacagcctgactttcaaggaggatattcaaaaggcacaggtgagcggccagggcgatagcctgcacgaacacatcgcaaatctggccggtagccctgccattaagaagggcatcctgcagacagtgaaggttgttgatgaactggtcaaggtgatgggtagacacaagcccgagaatattgtgatcgagatggctagagagaaccaaacaacacaaaagggacagaagaatagcagagaaagaatgaaaagaattgaggagggaatcaaggagctgggtagccagatcctgaaagaacaccctgtcgagaatacacaactgcaaaacgaaaagctgtacctgtactacctgcaaaatggcagagacatgtacgtggaccaagagctggatattaacagactgagcgactacgatgtcgaccacatcgtgcctcaaagcttcctgaaggatgacagcatcgacaataaagtgctgactagaagcgacaagaacagaggaaaaagcgacaacgtgcccagcgaggaagtggttaaaaagatgaagaactactggagacagctgctgaatgccaagctgatcacacaaagaaaattcgacaacctgaccaaagccgagagaggaggtctgagcgaactggacaaggctggattcattaagagacaactggttgaaaccagacagattacaaagcacgtggctcaaatcctggacagcagaatgaataccaaatatgacgagaacgacaaactgattagagaggtgaaggttattactctgaagagcaaactggtcagcgacttcagaaaggacttccaattctacaaggtgagagagatcaacaattaccaccacgcacacgacgcttacctgaacgctgtggtgggcacagctctgatcaaaaagtatccaaaactggaaagcgagtttgtgtacggtgactataaagtttatgatgtgagaaaaatgatcgctaagagcgagcaggagatcggaaaggctacagccaagtatttcttttacagcaacattatgaactttttcaagactgaaatcaccctggcaaacggtgagatcagaaaaagaccactgatcgaaacaaatggcgagacaggcgagatcgtgtgggataagggaagagacttcgctaccgttagaaaggttctgagcatgccacaggttaacattgtgaagaaaactgaggtgcagacaggaggtttcagcaaggagagcatcctgcctaagagaaacagcgataagctgattgcaagaaaaaaggattgggaccctaagaagtacggcggttttgacagccctactgtggcttacagcgtgctggtggtggctaaagtggagaagggcaaaagcaagaagctgaaaagcgtgaaggaactgctgggaattacaatcatggagagaagcagcttcgagaagaacccaatcgacttcctggaggctaagggatacaaggaagttaagaaggacctgatcatcaagctgcccaagtacagcctgttcgagctggaaaatggtagaaagagaatgctggctagcgctggtgagctgcagaagggaaatgaactggcactgcctagcaagtacgttaactttctgtatctggcaagccattacgagaaactgaaaggaagccccgaggacaatgagcagaaacaactgttcgtggaacagcacaaacactatctggacgagattatcgagcagatcagcgaatttagcaaaagagtgatcctggctgatgctaacctggataaagtcctgagcgcttacaacaaacatagagataagcctatcagagagcaggccgaaaacatcatccacctgttcacactgacaaacctgggcgctcctgccgctttcaagtactttgataccactattgatagaaagagatatactagcaccaaagaggtgctggacgccaccctgattcaccagagcattaccggactgtacgaaactagaatcgacctgagccaactgggaggagacaagagacccgctgcaactaaaaaggcaggtcaggccaaaaagaagaaa。

in another preferred embodiment, the agent comprises zCas9 mRNA, hey2 sgRNA and hey2 ^CKOIS A donor plasmid.

In another preferred embodiment, the hey sgRNA has the following nucleic acid sequence:

GGAAGGATAATGGTTGGGTgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttaaagct(SEQ ID NO.:11)。

in a fourth aspect of the invention there is provided a host cell comprising a construct as described in the first aspect, or having its genome integrated with one or more constructs as described in the first aspect.

In another preferred embodiment, the host cell comprises a zebra fish adult somatic cell, a zebra fish fetal somatic cell, or a zebra fish embryonic stem cell.

In another preferred embodiment, the host cell is a fertilized egg of a single cell stage of zebra fish.

In another preferred embodiment, the host cell is introduced into the cell with a construct according to the first aspect and/or a vector according to the second aspect by a method selected from the group consisting of: homologous recombination, microinjection, electroporation, liposome transfection, calcium phosphate precipitation, viral infection, or sperm vector.

In a fifth aspect of the invention, there is provided a method of preparing a transgenic cell in vitro comprising the steps of:

(i) Transfecting a cell with the construct of the first aspect and/or the vector of the second aspect such that the construct undergoes site-directed non-homologous recombination with a chromosome in the cell, thereby producing a transgenic cell.

In another preferred embodiment, in step (i), the method further comprises transfecting the cell with a site-specific cleavage construct to site-specific cleave the chromosome of the cell, wherein the site of site-specific cleavage is located at a chromosomal target segment of zebra fish.

In another preferred embodiment, the site of site-directed cleavage is located on chromosome 20 of zebra fish, specifically from position 39589569 to position 39591030 of nc_007131.

In another preferred embodiment, the method further comprises the step (ii): transgenic cells are selected for positive insertion of the nucleic acid construct and random deletion sequences do not affect expression of endogenous genes, including the target gene.

In another preferred embodiment, the transgenic cell prepared by the method expresses both the endogenous gene and the first exogenous gene.

In another preferred embodiment, HEY2 protein is normally expressed while EGFP is expressed in the transgenic cells prepared by the method.

In another preferred embodiment, the HEY2 protein expressed in the transgenic cells prepared by the method is the HEY2 protein ^zCKOIS The protein comprises a fusion product of an N-terminal bHLH domain, an Orange domain and a C-terminal protein-protein interaction YRPW ("Y") module with GSG-P2A-EGFP.

In another preferred embodiment, the transgene comprises a gene knock-in, a gene knock-out, or a combination thereof.

In a sixth aspect of the invention, there is provided a method of preparing a transgenic cell in vitro comprising the steps of:

(i) Transfecting a cell with the construct of the first aspect and/or the vector of the second aspect in the presence of Cre recombinase such that the construct undergoes site-directed non-homologous recombination with a chromosome in the cell, thereby producing a transgenic cell.

In another preferred embodiment, in step (i), further comprising: the cell is transfected with the site-directed cleavage construct, thereby site-directed cleavage of the chromosome of the cell, wherein the site of site-directed cleavage is located at the chromosomal target segment of the zebra fish.

In another preferred embodiment, the transgenic cell prepared by the method expresses both the first exogenous gene and the second exogenous gene.

In another preferred embodiment, the method produces transgenic cells in which the 3' last exon of the endogenous gene is deleted.

In another preferred embodiment, the endogenous gene transcription of the transgenic cell prepared by the method is truncated.

In another preferred embodiment, the EGFP and TagRFP are expressed simultaneously in the transgenic cells prepared by the method.

In another preferred embodiment, the transgenic cell prepared by the method expresses Hey2 ^zCKOIS-inv Proteins, said Hey2 ^zCKOIS-inv The protein is the product of fusion of P2A-TagRFP by the bHLH domain of the Hey2 protein.

In a seventh aspect of the present invention, there is provided a method of preparing a transgenic animal comprising the steps of:

(i) Transfecting a cell with the construct of the first aspect and/or the vector of the second aspect such that the construct undergoes site-directed recombination with a chromosome in the cell, thereby producing a transgenic cell, and wherein the site of site-directed cleavage is located at a chromosomal target segment of a zebra fish; and

(ii) Regenerating the obtained transgenic cells into an animal body, thereby obtaining a transgenic animal.

In another preferred embodiment, step (ii) comprises the steps of:

(ii-1) subjecting the obtained transgenic cells to somatic cloning as a nuclear donor, thereby obtaining transgenic animals.

In another preferred embodiment, the cell is a fertilized egg of a single cell stage of zebra fish.

In an eighth aspect of the present invention, there is provided a method of preparing a tissue-specific transgenic animal comprising the steps of:

(a) Preparing a transgenic animal F1 with a genome stable insert of the construct according to the first aspect according to the method of the seventh aspect;

(b) Hybridizing the transgenic animal F1 obtained in step (a) with an animal F2 that tissue-specifically expresses Cre recombinase; and

(c) Screening to obtain transgenic animals expressing the first exogenous gene and simultaneously expressing the second exogenous gene in a tissue-specific manner.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows hey2 ^zCKOIS A method for constructing zebra fish strain.

(A) Construction of intron-targeted hey2 using CRISPR/Cas9 systems ^zCKOIS Pattern diagram of zebra fish. Zebra fish hey gene has 5 exons, E4 and E5 representing exons 4 and 5, respectively. The sgRNA target sequence was red and the Protospacer Adjacent Motif (PAM) sequence was green. The left and right arm sequences of the Donor plasmid are indicated by the brown lines of the double arrow. The Donor plasmid and sgRNA, zCas9 mRNA are co-injected into zebra fish embryo to make hey2 ^zCKOIS Plasmid determinationIntegration into the hey site. The left arm of the Donor plasmid is 3300bp in length, including the original left arm and the inverted TagRFP box sequence in the genome. The right arm is 1107bp long. GSG-P2A is a glycine (glycine) -serine (serine) -glycine (glycine) -P2A sequence.

(B) Pair hey2 ^zCKOIS The knocked-in F1 generation genomic DNA was subjected to PCR analysis. The 3.2kb length of DNA band was amplified using the F1 and R1 primers, and the 5.3kb length of DNA band was amplified using the F1 and R2 primers. The two strips are only hey2 ^zCKOIS Embryos are present but not in the Wild Type (WT) group. Specific positions of the primers F1, R1, F2, and R2 are shown in (A).

(C) The left channel shows analysis hey2 using RT-PCR technique ^zCKOIS cDNA Synthesis of F1 embryo. The 1.5kb band amplified by the F1 and R1 primers appears only at hey2 ^zCKOIS And (3) in the embryo. The right channel shows the result of RT-PCR detection using primers that bind to the hey2 coding sequence, showing a 0.3kb band that is consistent with the hey coding sequence.

(D)hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the 1.5 day confocal projection images (side view) of Ki (GFAP-TagBFP) embryos showing EGFP expression in TagBFP-labeled brain glial cells. Ki (GFAP-TagBFP) is a knock-in line that allows the specific expression of the TagBFP protein in glial cells. White arrow, midbrain; arrow, forebrain. Scale bar: 100 μm.

(E) Confocal projection images (side view) of Ki (GFAP-TagBFP); at hey2 ^zCKOIS In embryos (3.5 days), EGFP is expressed in glial cells of the spinal cord (white arrow). Cyan arrow, nonspecific signal on skin. Scale bar: 100 μm.

(F and G) are hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the Confocal projection images of Tg (flk 1: ras-mCherry) 3.5 day embryo; (F) Expression of EGFP in the basal cerebral arterial loop (BCA) and the posterior major venous junction (PCS) (dorsal view). Top left: an EGFP signal; top right: overlapping signals (EGFP/mCherry). The areas of a and b outlined in the figure on the right side of the top are shown enlarged in the bottom channel. White arrows, BCA and PCS; white arrows, choroidal plexus (CVP). (G) In zebra fish embryo In the trunk, EGFP is expressed in the Dorsal Aorta (DA) but not in the posterior main vein (PCV) (side view). Internode arterial segment vessels (aISVs) extending from DA exhibit more EGFP signals than internode venous vessels (vISVs) extending from PCV. Top left: an EGFP signal; top right: overlapping signals (EGFP/mCherry). The areas of a and b outlined in the figure on the right side of the top are shown enlarged in the bottom channel. White arrows, DA and aISVs; white arrows, PCV and vISVs. Cyan arrow, nonspecific signal on skin. Scale bar: 100 μm.

FIG. 2 demonstrates hey2 ^zCKOIS-inv Is a loss-of-function allele.

(A)hey2 ^zCKOIS Allelic and posttranslational hey2 ^zCKOIS Schematic representation of the protein. Endogenous Hey2 proteins include an N-terminal bHLH domain, an Orange domain, and a near C-terminal protein-protein interaction YRPW ("Y") module. Hey2 ^zCKOIS The protein is a fusion product of wild-type (WT) Hey2 protein and GSG-P2A-EGFP.

(B) Cre enzyme induced hey2 ^zCKOIS Inversion of the allele component yields hey2 ^zCKOIS-inv Schematic representation of alleles. Hey2 ^zCKOIS-inv The protein is P2A-TagRFP fused by bHLH domain of Hey2 protein.

(C) PCR detection hey2 ^zCKOIS-inv Is a genome that is inverted. A band of 2.8kb in length was present only in hey2 ^zCKOIS-inv In the absence of WT or hey2 ^zCKOIS And (3) in the embryo.

(D) Left channel, RT-PCR detection hey2 ^zCKOIS-inv Is a transcription of (a). hey2 ^zCKOIS-inv The band of 0.9kb in length was amplified, WT and hey2 ^zCKOIS None of the embryos amplified. On the right hand side, RT-PCR controls were performed using primers that bind to the hey2 coding sequence, each set having a 0.3kb band.

(E)hey2 ^zCKOIS-inv The method comprises the steps of carrying out a first treatment on the surface of the 1.5 day embryo confocal projection images (side view) of Ki (GFAP-TagBFP), tagRFP was able to be expressed in glial cells. The enlarged view of the box area is shown on the right. White arrow, midbrain; arrow, forebrain.

Scale bar: 100 μm.

(F)hey2 ^{zCKOIS/zCKOIS-inv} Confocal projection images of embryo trunk for 2.5 days (side view). Red fluorescence is represented by hey2 ^zCKOIS-inv Hey2 (truncated) -P2A-TagRFP encoded translation in the allele, green fluorescence was translated by WT Hey2-GSG-P2A-EGFP encoded translation. Cyan arrow, nonspecific signal on skin. Scale bar: 100 μm.

(G) Hey2 2.5 days ^{zCKOIS/zCKOIS-inv} Confocal projection images of DA on embryonic torso showed two HSCs sprouting from DA. Scale bar: 50 μm.

(H) Top channel: bright field image display homozygote hey2 ^zCKOIS-inv / ^zCKOIS-inv There is severe pericardial edema. The bottom channel: heterozygote hey2 ^zCKOIS-inv No significant edema was observed at 3.5 days. Left channel: bright Field (BF) and TagRFP. Right side channel: tagRFP. Scale bar: 500 μm.

(I) In embryos with different genetic backgrounds, the normal proportion of tail circulation is shown. The numbers in the figure are the total number of embryos of the different groups for 3.5 days.

FIG. 3 shows specific knockdown of hey2 gene in endothelial cells.

(A) Schematic representation of hey2 in specific knockdown endothelial cells. By combining the pure Ki (flk 1-P2A-Cre) line with hey2 ^zCKOIS The strain is mated, and Hey2 protein in ECs of the obtained offspring is specifically destroyed. Hey2 in non-ECs which do not express Cre ^zCKOIS Is not reversed, so that the Hey2 protein is expressed normally.

(B) RT-PCR experiments with cDNA, detection hey2 ^zCKOIS And hey2 ^zCKOIS-inv Is a transcription situation of (a). The left channel is F1 and R2 primer to amplify the band with the size of 1.5kb of the target gene, hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the Ki (flk 1-P2A-Cre) and hey2 ^zCKOIS The embryos of the WT group were amplified, whereas the embryos of the WT group were not amplified. The intermediate channel, a 0.9kb band amplified by the F1 and F2 primers, was present only in hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the Ki (flk 1-P2A-Cre) embryo. On the right hand side, RT-PCR controls were performed using primers that bind to the hey2 coding sequence, each set having a 0.3kb band.

(C) 3.5 days hey2 ^{zCKOIS/zCKOIS} The method comprises the steps of carrying out a first treatment on the surface of the Confocal projection images (side view) of trunk vessels in Ki (flk 1-P2A-Cre) embryos. In DA and aISVs, the inversion of the Cre-induced module initiates hey2 ^zCKOIS In which EGFP expression is hardly subtracted (indicated by arrows and dashed lines). The EGFP signal is on the left of the top; on the right top, overlap signal (EGFP/TagRFP). The outline in the figure is an enlarged area. Cyan arrow, nonspecific signal on skin. Scale bar: 100 μm.

(D) In embryos with different genetic backgrounds, the normal proportion of tail circulation is shown. The numbers in the figure are the total number of embryos of the different groups for 3.5 days.

FIG. 4 shows the sequencing of the fish line hey2 ^zCKOIS Genomic and transcriptional analysis was performed. (A) Pair hey2 ^zCKOIS The F1 generation of the knockin line was sequenced at the 5' segment of the Donor plasmid integration site. PAM and sgRNA target sequences are shown as green and red, respectively. At integration hey2 ^zCKOIS Introns near the target region of the sgRNA of the Donor plasmid had 894bp base deletions. (B) hey2 ^zCKOIS The transcribed cDNA sequence showed that EGFP was directly linked in-frame to exon5 of hey 2.

FIG. 5 shows the sequencing of the fish line hey2 ^zCKOIS-inv Genomic and transcriptional analysis was performed. (A) Pair hey2 ^zCKOIS ^-inv The F1 generation of the knockin line was sequenced at the 5' segment of the Donor plasmid integration site. PAM and sgRNA target sequences are shown as green and red, respectively. Introns near the sgRNA target region and hey2 ^zCKOIS There was the same 894bp deletion in the line. (B) hey2 ^zCKOIS-inv The transcribed cDNA sequence showed that TagRFP was ligated to exon4 of hey.

FIG. 6 shows hey2 ^{zCKOIS/zCKOIS-inv} Confocal projection images of embryos. (A) co-locating the map. Example ruler: 100 μm. (B) At hey2 ^{zCKOIS/zCKOIS-inv} Embryo for 2.5 days, EGFP (from hey2 ^zCKOIS Encoded) and TagRFP (from hey2 ^zCKOIS-inv Code) co-localization in fluorescence signal gill cap artery (ORA). Scale bar: 50 μm.

FIG. 7 shows hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the Genotyping of Ki (flk 1-P2A-Cre) fish line. Left picture, basePCR analysis of genomic DNA showed hey2 ^zCKOIS And hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the The Ki (flk 1-P2A-Cre) group had a 3.2kb band, whereas the WT group did not. Right side view, only hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the Ki (flk 1-P2A-Cre) group and positive control hey2 ^zCKOIS-inv The group had a 2.8kb sized band, the WT group and hey2 ^zCKOIS The group does not exist.

FIG. 8 shows hey2 ^{zCKOIS/zCKOIS} Confocal projection images of strain trunk. When Cre is absent, EGFP is expressed in DA (indicated by an arrow), and no red fluorescent signal of TagRFP is detected. Asterisks, nonspecific signal on yolk sac. Cyan arrow, nonspecific signal on skin. Scale bar: 100 μm.

FIG. 9 shows the verification of Ki (flk 1-P2A-Cre) fish lines.

Ki (flk 1-P2A-Cre); tg (bacin 2: loxP-STOP-loxP-DsRedEx); confocal projection images on the torso of 3.5 day embryos were Tg (flk 1: EGFP). Red represents bacin 2, dsRedEx. EGFP represents flk1 EGFP. Scale bar: 100 μm.

Detailed Description

The inventor establishes a high-efficiency and tissue-specific zebra fish gene knockout method by inserting an exogenous gene expression cassette between the left arm and the right arm of a donor plasmid and reversely constructing a section of DNA sequence element containing two LoxP sites in the left arm intron sequence, namely an inverted second exogenous gene expression cassette. In the absence of Cre protein expression, the endogenous gene and the first exogenous gene are normally expressed with complete structure and function. In the case of expressing Cre protein, the DNA sequence elements of two LoxP sites are inverted by Cre and bind to the previous exon of the gene through the splice acceptor, disrupting the structure of the gene, and the mutated gene also expresses the second exogenous gene. The present invention has been completed on the basis of this finding.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of a reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a well known method to those skilled in the art.

As used herein, the terms "gene inactivation", "gene knockout" and "gene knockdown" are used interchangeably to refer to a substantial reduction or even complete loss of expression and/or activity of a gene of interest by disruption, knocking out, or the like of that gene.

As used herein, the term "hey2 ^zCKOIS Donor plasmid "," Donor plasmid "may be interchanged.

CRISPR/Cas system

The CRISPR/Cas system (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is an acquired immune defense mechanism in prokaryotes against the invasion of foreign genes. Are evolving from bacteria and archaea in the process of protecting against the invasion of foreign viruses and phages. The system is capable of integrating a DNA fragment of a foreign invaded host into a CRISPR site and then guiding Cas endonuclease cleavage of the foreign DNA sequence by corresponding CRISPR RNAs (crRNAs) to resist virus or phage invasion. The CRISPR/Cas gene cluster consists of a series of coding genes for Cas proteins (Cas 1, cas2, cas4 and effector proteins such as Cas9, cpf1, etc.) and a section of CRISPR sequence,

CRISPR sequences consist of a leader, a number of short and conserved repeat regions (repeats) and spacers. The repeat sequence region contains a palindromic sequence capable of forming a hairpin structure. The spacer is an exogenous DNA sequence captured by the host. These captured exogenous DNA sequences correspond to a "blacklist" of the immune system, and when these exogenous genetic material invade the host again, the bacteria begin to transcribe CRISPR, forming the primary transcript pre-crRNA, and then the ribonuclease or Cas protein cleaves at the site of the repeat sequence to form mature crRNA, forming ribonucleoprotein complexes with specific CRISPR effector proteins, recognizing and cleaving exogenous DNA that can be complementarily paired with crRNA, causing double strand breaks, initiating self-repair of the host cell.

CRISPR is classified into type 2, type 5, 16 subtypes in total, depending on the composition of Cas gene and the number of effector proteins. Class 1 is a CRISPR/Cas system that utilizes multiple effector protein complexes to interfere with a target gene, including types i, iii, and iv; class 2 is a CRISPR/Cas system that utilizes a single effector protein to interfere with a target gene, including types ii and v. The most widely studied and utilized type 2 is the CRISPR/Cas9 system. The system successfully achieved gene editing in mammalian cells in 2013. Type ii systems can use a single Cas9 nuclease to precisely and fully cleave DNA target sites through the guidance of crrnas. The system is simple to operate, short in experimental period, high in efficiency and widely applicable to multiple species. The system requires the design of a specific guide RNA, sgRNA (single guide RNA), the sgRNA sequence design is about 20nt of the PAM (NGG) region in the genomic sequence. Under the guidance of sgRNA, cas9 protein can realize site-directed cleavage on genome, cause DNA double strand break, activate two repair mechanisms of Non-homologous end joining (Non-homologous end joining, NHEJ) or homologous recombination (Homologous recombination, HR) of cells, thereby realizing knockout of gene, random fragment deletion or insertion, or repair by using a specific template, thereby realizing permanent modification on genome.

As used herein, the terms "single guide RNA", "sgRNA" are interchangeable.

In a preferred embodiment, the Cas9mRNA sequence is zCas9mRNA optimized for zebra fish codon bias, the nucleic acid sequence of which is set forth in SEQ ID No. 10.

Cre-LoxP system

The full length 1029bp (EMBL database accession number X03453) of Cre recombinase gene coding region sequence codes for a 38kDa protein. Cre recombinase is a monomeric protein consisting of 343 amino acids. Belongs to lambda Int enzyme super gene family, has catalytic activity, and can recognize specific DNA sequence, i.e. loxP site, similar to restriction enzyme, so that the gene sequence between loxP sites can be deleted or recombined.

The loxP (loxP 1) sequence is derived from P1 phage, and consists of two 13bp inverted repeats and an intermediate 8bp sequence, and the 8bp sequence also determines the direction of the loxP. The Cre enzyme is covalently bound to DNA during the catalytic DNA strand exchange process, and the 13bp inverted repeat is the Cre enzyme binding domain.

The loxP site used in the present invention includes a wild-type loxP site and a mutant loxP site, and the common mutant loxP site includes: lox511, lox5171, lox2272, loxm2, loxm3, loxm7, and loxm11.

In a preferred embodiment, the two loxP sites used in the present invention are a wild-type loxP site and a mutant lox5171 site, wherein the wild-type loxP site is shown in SEQ ID NO. 4, and the mutant lox5171 site is shown in SEQ ID NO. 5.

Nucleic acid constructs

The invention also provides a construct according to the first aspect of the invention.

The various elements used in the constructs of the invention are known in the art and thus the skilled artisan can obtain the corresponding elements by conventional methods, such as PCR, total artificial chemical synthesis, and digestion, and then ligate them together by well known DNA ligation techniques to form the constructs of the invention.

The constructs of the invention are inserted into exogenous vectors, particularly vectors suitable for manipulation by transgenic animals, to form vectors of the invention.

The vector of the present invention is transformed into a host cell to mediate the integration of the vector of the present invention into the chromosome of the host cell, thereby producing a transgenic cell.

As used herein, "exogenous gene" refers to an exogenous DNA molecule that acts in a stepwise manner. The foreign genes useful in the present application are not particularly limited, and include various foreign genes commonly used in the field of transgenic animals. Representative examples include (but are not limited to): red fluorescent protein gene, green fluorescent protein gene, lysozyme gene, salmon calcitonin gene, lactoferrin, serum albumin gene, or the like.

In a preferred embodiment, two "exogenous genes," TagRFP and EGFP, are used in the present invention. The amino acid sequence of the TagRFP sequence is shown as SEQ ID NO. 8, and the EGFP sequence is shown as SEQ ID NO. 3.

As used herein, "selectable marker gene" refers to a gene used in a transgenic process to screen transgenic cells or transgenic animals, and selectable marker genes useful in the present application are not particularly limited, including various selectable marker genes commonly used in the transgenic art, representative examples including (but not limited to): neomycin gene, or puromycin resistance gene.

As used herein, the term "expression cassette" refers to a polynucleotide sequence that contains the gene to be expressed and the sequence components that express the desired elements. For example, in the present invention, the term "selectable marker expression cassette" refers to a polynucleotide sequence that contains the sequence encoding the selectable marker as well as sequence components that express the desired elements. The components required for expression include a promoter and polyadenylation signal sequences. In addition, the selectable marker expression cassette may or may not contain other sequences including (but not limited to): enhancers, secretion signal peptide sequences, and the like.

In a preferred embodiment, two self-cleaving element P2A sequences and a GSG-P2A sequence are used in the present invention. The nucleic acid sequence of the P2A sequence is shown as SEQ ID NO. 6, and the nucleic acid sequence of the GSG-P2A is glycine (glycine) -serine (serine) -glycine (glycine) -P2A sequence.

In the present invention, the promoter suitable for the foreign gene expression cassette and the selectable marker gene expression cassette may be any one of common promoters, which may be a constitutive promoter or an inducible promoter. Preferably, the promoter is a constitutive strong promoter, such as bovine beta-lactoglobulin promoter and other promoters suitable for eukaryotic expression, and the BGHpA signal sequence is shown in SEQ ID NO. 7.

Hey2 gene

The Hey2 gene, known as hes related family bHLH transcription factor, carries the YRPW motif 2 (hes-related family bHLH transcription factor with YRPW motif 2), and is involved in circulatory system development. The zebra fish hey2 gene is positioned on chromosome 20, and has 5 exons, the full length of the nucleic acid sequence is 7524bp, and the GenBank accession number is NC_007131; the amino acid sequence of the expressed protein is 324aa, and the accession number is NP-571697.2.

The invention utilizes CRISPR/Cas9 system to construct hey2 ^zCKOIS The zebra fish strain provides a sgRNA targeting a segment of an intron between the 4 th and 5 th exons of a zebra fish hey gene (the target sequence is shown as SEQ ID NO. 2, PAM is AGG), and the nucleic acid sequence of the hey sgRNA is shown as SEQ ID NO. 11.

In the invention, a hey2 is also provided ^zCKOIS Donor plasmid (Donor plasmid) whose left and right arm sequences are shown in fig. 1A. Wherein the Donor plasmid has a left arm length of 3300bp and comprises the original left arm, inverted DNA module in the genome. The inverted DNA module has TagRFP box tag sequence, cleavage acceptor and 2 sets of loxP sites (loxP, lox 5171). In a preferred embodiment, the inverted DNA module is linked to the hey left arm sequence by a ClaI cleavage site. The original left arm contained exon 5E 5 of hey2 and a stretch of intronic sequence containing the sgRNA target sequence. The Donor plasmid is identical to the original right arm, 1107bp long, and contains the 3 'gene spacer region of the whole 3' UTR sequence of the target gene. hey2 ^zCKOIS GSG-P2A self-cleavage sequence and EGFP tag fragment are also inserted between the left and right arms of the donor plasmid, and the GSG-P2A is glycine(glycine) -serine (serine) -glycine (glycine) -p2a sequence. In a preferred embodiment, the left and right arms are linked to the 5 'and 3' ends of the GSG-P2A-EGFP fragment in the T-GSG-P2A-EGFP vector, respectively, to give hey2 ^zCKOIS A donor plasmid having the sequence set forth in SEQ ID No.: 9.

Application of

In one embodiment of the invention, there is provided an agent comprising zCas9 mRNA, hey2 sgRNA and hey2 ^CKOIS A donor plasmid. In a preferred embodiment, the reagent contains 800 ng/. Mu.l zCas9 mRNA,80 ng/. Mu.l sgRNA and 15 ng/. Mu.l donor plasmid. In a preferred embodiment, the Cas9 mRNA sequence is zCas9 mRNA optimized for zebra fish codon bias, the nucleic acid sequence of which is set forth in SEQ ID No. 10.

In one embodiment of the invention, there is also provided a hey2 ^zCKOIS Protein, hey2 ^zCKOIS The protein is a fusion product of wild-type (WT) Hey2 protein and GSG-P2A-EGFP.

In one embodiment of the invention, there is also provided a method of obtaining hey2 ^zCKOIS A method of fish-based, comprising the steps of: (a) Providing a single-cell fertilized egg, and injecting zCas9 mRNA, hey2 sgRNA and hey2 into the single-cell fertilized egg ^CKOIS Culturing the donor plasmid reagent for several days to obtain transgenic adult fish; (b) Crossing the transgenic adult fish obtained in step (a) with wild zebra fish, identifying genomic DNA of the filial generation, and screening to obtain hey2 ^zCKOIS And (5) fish line. In a preferred embodiment, the hey2 ^zCKOIS Expression of the fish line hey2 ^zCKOIS And (3) protein. In a preferred embodiment, the hey2 ^zCKOIS The cDNA sequence transcribed from the fish line shows that EGFP is directly linked in frame to exon5 of hey. In a preferred embodiment, the hey2 ^zCKOIS Simultaneous expression of EGFP and hey2 in cells of fish lines ^zCKOIS And (3) protein.

In one embodiment of the invention, there is also provided a hey2 ^zCKOIS-inv Protein, hey2 ^zCKOIS-inv The protein is a fusion product of fusion of P2A-TagRFP by the bHLH domain of the Hey2 protein.

In one embodiment of the present invention, there is also providedObtained hey2 ^zCKOIS-inv A method of fish-based, comprising the steps of at hey2 ^zCKOIS The Cre recombinase is simultaneously expressed in cells of the fish line. In a preferred embodiment, the hey2 ^zCKOIS-inv Expression of the fish line hey2 ^zCKOIS-inv And (3) protein. In a preferred embodiment, the hey2 ^zCKOIS-inv The cDNA sequence transcribed from the fish line shows that TagRFP is linked to exon4 of hey. In a preferred embodiment, the hey2 ^zCKOIS Simultaneous expression of TagRFP and hey2 in cells ^zCKOIS-inv And (3) protein. In a preferred embodiment, the obtaining hey2 ^zCKOIS-inv A method of fish line comprising adding hey2 ^zCKOIS The fish line was crossed with the Cre-specifically expressed knock-in line. In a preferred embodiment, the Cre-specific expressed knock-in line comprises the Ki (flk 1-p 22-Cre) line, the Ki (GFAP-TagBFP) line. The nucleic acid sequence of the Cre recombinase mRNA is shown in SEQ ID NO. 12.

The main advantages of the invention include:

1. The invention provides a tissue-specific zebra fish gene knockout method, which uses a non-homologous recombination technology, and the knockout efficiency is obviously improved compared with homologous recombination.

2. The method has the outstanding characteristics that a section of exogenous gene can be knocked in a whole body, and the function of a target gene in a nonspecific tissue is not affected; the target gene can be knocked out in a tissue specificity mode in a non-specific tissue, a section of exogenous gene can be knocked in a fixed point specificity mode, multiple transgenic effects can be obtained through a one-step method, and the transgenic efficiency is greatly improved.

3. The invention can further simplify the screening step, and the exogenous gene is designed to express fluorescent protein, so that the target transgenic zebra fish can be obtained simply and conveniently by screening embryos with specific fluorescent markers intuitively.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Materials and methods

Flk1 gene and Ki (Flk 1-p 22-cre) line

The Flk1 gene, which is called kinase insert region receptor (kinase insert domain receptor like), is located on chromosome 14 and contains 30 exons, the full length of the nucleic acid sequence 61184bp, and the GenBank accession number is NC_007425; the amino acid sequence of the expressed protein is 1300aa, and the accession number is XP_009289417.

The Ki (flk 1-p 22-cre) line used in the present invention was generated by the CRISPR/cas9 mediated KI method, and was accomplished as described in the prior patent (patent No. 201510075155.6). Briefly, the EGFP sequence in the flk1-P2A-EGFP donor plasmid was replaced with Cre to give the flk1-P2A-Cre plasmid.

Tg(bactin2:loxP-stop-loxP-dsred-express) ^sd5 Fish series

The transgenic fish was given from the professor David river, the university of california, san diego division biology department of science. The method is mainly used for verifying the cleavage efficiency of Cre protein expressed by vascular endothelial cells in Ki (flk 1-p 22-Cre) fish line. The transgenic fish line is driven by the bacin 2 promoter to express a DNA sequence containing loxP-stop-loxP-dsred-express. loxP is the loxP site; stop is the stop codon sequence; dsred-express is a red fluorescent protein sequence. The transgenic fish line does not express dsred-express red fluorescent protein in the absence of Cre protein; if Cre protein is present, stop sequence is cleaved and dsred-express red fluorescent protein is expressed.

Ki (GFAP-TagBFP) fish line

Ki (GFAP-TagBFP) fish line was accomplished as described in the prior art patent (patent No. 201510075155.6). The simplified description of the procedure as GFAP-TagBFP donor plasmid is accomplished by replacing the EGFP sequence in the GFAP-EGFP donor with TagBFP.

Zebra fish breeding

Adult zebra fish are cultured in an aquatic animal culture system of Beijing Alsheng company at a water temperature of 28 ℃ and a pH of 7-8 in a light/dark environment with a light cycle of 14 hours. Embryos were raised in 10% Hank's solution. The solution composition was as follows (millimoles): 140NaCl,5.4KCl,0.25Na ₂ HPO ₄ ，0.44KH ₂ PO ₄ ，1.3CaCl ₂ ，1.0MgSO ₄ And 4.2NaHCO ₃ (pH 7.2)。

Example 1sgRNA,zCas9 mRNA and Cre mRNA Synthesis

The zCas 9-expressing plasmid pGH-T7-zCas9 was obtained from the university of Beijing college of student's university of Ming. After linearizing this plasmid with Xba I endonuclease, zCas9 mRNA was obtained by transcription purification using mMACHINE T7 Ultra kit (Ambion) kit. The Cre coding sequence was amplified from the Cre plasmid using a primer containing the T7 promoter. Cre mRNA was synthesized from the same T7 Ultra kit.

The zCas9 mRNA has the sequence shown in SEQ ID NO. 10 and Cre mRNA has the sequence shown in SEQ ID NO. 12.

The targeted DNA sequences used to prepare the different sgrnas are shown below:

hey2	GGAAGGATAATGGTTGGGT	located on the sense strand	SEQ ID No.:13
				gfap	GTGCGCAACACATAGCACCA	Located on the antisense strand	SEQ ID No.:14
flk1	TCTGGTTTGGAAGGACACAG	Located on the sense strand	SEQ ID No.:15

The above sgRNA sequences were cloned into the BbsI site of the PT7-sgRNA plasmid (obtained from the university of Beijing university, proc. Natl. Acad. Sci.) and transcribed in vitro by the MAXIscript T7 Kit (Ambion) and used as a plasmid for the mirVana ^TM miRNA Isolation Kit (Ambion) kit was recovered to obtain sgRNA. The sequence of the sgRNA is exemplified by hey2 sgRNA, and the obtained hey sgRNA has a nucleic acid sequence shown in SEQ ID NO. 11.

EXAMPLE 2 construction hey2 ^CKOIS ' Zebra fish strain and verification

2.1 GSG-P2A-EGFP fragment was ligated to a PMD-19-T vector (purchased from Takara) by T-A cloning method to form a T-GSG-P2A-EGFP vector. The left and right arms of hey were amplified from wild-type zebra fish genomic DNA using KOD-PLUS Neo DNA polymerase. Then, left and right arms were ligated to the 5 'and 3' ends of the GSG-P2A-EGFP fragment in the T-GSG-P2A-EGFP vector, respectively. The whole gene synthesizes an inverted DNA module with loxP site, splice acceptor, tagRFP and BGHpA. The resulting inverted DNA module was 1.8kb in size and was digested with ClaI cleavage sites (AT ^▼ CTAG) (ligation to hey2 left arm sequence gives hey2 ^CKOIS A donor plasmid. Finally, zCas9 mRNA, hey sgRNA and hey2 ^CKOIS Donor plasmids were co-injected into single cell stage fertilized eggs. Each embryo was injected with 1nl of solution containing 800 ng/. Mu.l zCas9 mRNA,80 ng/. Mu.l sgRNA and 15 ng/. Mu.l donor plasmid. The left and right arm sequences of the donor plasmid were amplified from adult wild type AB zebra fish genomic DNA using the following primers:

1) Left arm amplification primers:

F(l)	5′-CGAGGTACCCACTCGTCGACAAAACTAGGG-3′	SEQ ID No.:16
			R(l)	5′-CGAGGATCCAAACGCTCCCACTTCAGTTC-3′	SEQ ID No.:17

2) Right arm amplification primers:

F(r)	5′-CGAACCGGTTAAATGTTGGATTTAAATGT-3′	SEQ ID No.:18
			R(r)	5′-CGACTGCAGTAGGGTTTTAGCAGGCACCG-3′	SEQ ID No.:19

2.2 for screening hey2 ^CKOIS Adult fish were hybridized to wild-type (WT) zebra fish, genomic DNA (dpf) was extracted 3 days after fertilization, and identified by PCR detection.

Zebra fish embryo total RNA (Invitrogen, 15596018) at 1.5dpf and 3.5dpf was extracted using TRIzol reagent according to the instructions. The total RNA extracted was PrimeScript ^TM RT Master Mix (Takara, RR 036A) generated first-strand cDNA.

hey2 ^zCKOIS And hey2 ^zCKOIS-inv Information of primer F1\R1 of PCR of genomic DNA and primer F2\R2 of RT-PCR analysis of cDNA:

F1	5′-GATCTGCCAAGTTGGAGAAAGC-3′	SEQ ID No.:20
			F2	5′-TCAATTAAGTTTGTGCCCCAGT-3′	SEQ ID No.:21
R1	5′-CACCGTGAACAACCACCACT-3′	SEQ ID No.:22
			R2	5′-CTTGTACAGCTCGTCCATGCC-3′	SEQ ID No.:23

primer F3/R3 for RT-PCR analysis of hey2 cDNA:

F3	5′-ATGAAGCGGCCCTGTGAGGA-3′	SEQ ID No.:24
			R3	5′-CTTTTCCTCCTGTGGCCTGAA-3′	SEQ ID No.:25

2.3 for screening hey2 ^CKOIS In the fish line, adult fish are hybridized with Wild Type (WT) zebra fish, genomic DNA (dpf) is extracted 3 days after fertilization, and the detection and identification are further carried out by using an imaging technology.

Confocal imaging sequential fluorescence images were taken in the Z-axis direction in optical slices using a FN1 confocal microscope (Nikon, japan) with a 25-fold (n.a., 1.1) or 10-fold (n.a., 0.3) water mirror. The resolution of all pictures is 1024×1024 or 512×512. Later the structural morphology was reconstructed by ImageJ software (NIH).

Results:

the probability of the screened foundation which can be stably inherited to the next generation of F1 and has the forward insertion direction is as follows: 2/21. That is, 21 microinjected F0 zebra fish were screened, two of which were screened. However, the sequence of the donor plasmid was inserted into the genome of zebra fish by homologous recombination, but no founder was selected, and even if 50 microinjected F0 zebra fish were selected, no founder that could be stably inherited to the next generation was obtained. The proposal provided by the invention can efficiently and stably edit the zebra fish genome at fixed points.

As shown in FIGS. 1B-C, the PCR electrophoresis strip demonstrated successful construction of hey2 ^zCKOIS Zebra fish model, i.e. hey2 ^zCKOIS Cell with engineered hey2 inserted therein ^zCKOIS The left arm of the donor plasmid and the GSG-P2A-EGFP structure express hey2 ^zCKOIS And (3) protein. As shown in FIGS. 2C-D, the PCR and RT-PCR electrophoresis bands demonstrated successful construction of hey2 ^zCKOIS-inv Zebra fish model, i.e. hey2 ^zCKOIS-inv The cells have been inserted with P2A-TagRFP, i.e. hey2 is expressed ^zCKOIS-inv And (3) protein. As shown in FIG. 3B, RT-PCR electrophoresis bands demonstrated that EGFP-containing fragments were inserted into embryonic cells, but only hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the Cre enzyme-induced P2A-TagRFP fragment turnover, i.e., cre enzyme action, occurs in Ki (flk 1-P2A-Cre) line embryo cells.

As shown in FIGS. 4 and 5, the fish line hey2 was sequenced ^zCKOIS 、hey2 ^zCKOIS-inv Genome and transcription were verified, demonstrating the introduction of hey2 ^zCKOIS Donor plasmid resulted in integration hey2 ^zCKOIS Introns near the target region of the sgRNA of the Donor plasmid had 894bp base deletions. The deletion of the sequence is caused by the randomness of the repair of the genome DNA after the Cas9/sgRNA is cut, so that the sequence addition and deletion caused by the deletion are random, and generally vary from a few base pairs to hundreds of base pairs.

Immunofluorescence marker imaging as shown in FIGS. 1D-G, FIGS. 2E-I, and FIGS. 3C-D also demonstrate the conclusion that hey2 was successfully constructed ^zCKOIS Zebra fish model, and hey2 ^zCKOIS-inv Is a loss-of-function allele. As shown in FIG. 6, pair hey2 ^{zCKOIS/zCKOIS-inv} Confocal projection images of embryos showed embryos for 2.5 days, EGFP (from hey2 ^zCKOIS Encoded) and TagRFP (from hey2 ^zCKOIS ^-inv Code) co-localization in fluorescence signal gill cap artery (ORA). As shown in FIG. 8, pair hey2 ^{zCKOIS/zCKOIS-inv} Confocal projection images of the torso showed embryos for 2.5 days, EGFP was expressed in DA (indicated by the arrow) when Cre was absent, and red fluorescent signal of TagRFP was not detected (asterisk, non-specific signal on yolk sac; cyan arrow, non-specific signal on skin) also demonstrated hey2 ^zCKOIS-inv Is a loss-of-function allele.

Example 3 construction of a knock-in line Ki with Cre-specific expression (flk 1-P2A-Cre)

Ki (flk 1-p 22-cre) fish lines were generated by the CRISPR/cas9 mediated KI method, and were accomplished as described in the prior patent (patent No. 201510075155.6). Briefly, the EGFP sequence in the flk1-P2A-EGFP donor plasmid was replaced with Cre to give the flk1-P2A-Cre plasmid. Injecting 80pg of flk1gRNA and 15pg of flk1 donor plasmid into zebra fish embryos in 1nl of solution containing 800pg of zCas9 mRNASingle cell phase. These embryos were grown to adulthood and subjected to primary screening. To screen Ki (flk 1-P2A-Cre) lines, adult fish were hybridized to AB wild-type zebra fish, genomic DNA (dpf) was extracted 1 day after fertilization, and identified by PCR. The fish line was then combined with Tg (bacin 2: loxP-stop-loxP-dsred-express) ^sd5 Fish lines mate to determine Cre-mediated deletion of loxP sites in blood vessels.

Results: hey2 ^zCKOIS The method comprises the steps of carrying out a first treatment on the surface of the The PCR analysis of the genotype of the Ki (flk 1-P2A-Cre) fish line is shown in FIG. 7. Ki (flk 1-P2A-Cre) 3.5 days; tg (bacin 2: loxP-STOP-loxP-DsRedEx); the results of confocal projection images on the torso of Tg (flk 1: EGFP) embryos are shown in FIG. 9. The Cre-mediated deletion of loxP site in blood vessels in Ki (flk 1-P2A-Cre) fish line was demonstrated.

Discussion of the invention

The application of the current gene operation in zebra fish is just started. The limiting factors are mainly that in vitro culture and zebra fish embryo stem cell operation technologies are not available, and the generation of the zebra fish and the generation of the chimeric zebra fish cannot be realized at the present stage. Therefore, it is not possible to establish a zebra fish gene knock-in line by using the homologous recombination in vitro targeting method commonly used in mice.

Meanwhile, since the homologous recombination efficiency at the cell background level is very low, if only targeting vectors comprising left and right homology arms are injected into fertilized eggs, it is difficult to achieve gene knock-in at the in-vivo level. However, if the double-stranded DNA breaks generated by the genome are used (double strands break, DSB), the efficiency of homologous recombination can be increased by several orders of magnitude. The efficient cleavage of DNA by Cas9 system to generate DSBs provides a simpler approach for gene knock-in. The us Jaenisch teaches that the laboratory first uses Cas9 system in mice to achieve site-directed insertion of large fragments of DNA. They co-injected Cas9 mRNA, sgRNA and template plasmid into fertilized eggs of mice in one cell stage, and insert large-fragment reporter genes into 3' ends of Nanog and Oct4 genes by homologous recombination mechanism, successfully achieved protein labeling of embryonic stem cells and targeted gene expression (Yang, h.et al cell 154, 1370-1379).

However, this strategy is not yet applied to zebra fish efficiently to achieve the knocking-in of double LoxP sites, one of the important reasons is that zebra fish fertilized eggs stay for a cell period very short, only ten minutes (mice have hours), so that a short time window is difficult to achieve efficient mediated homologous recombination, and thus the integration efficiency is low.

Another important reason is that the genomic background of zebra fish is relatively complex, the homozygosity of the genomic sequence of the inbred line is insufficient, and it is difficult to clone long fragment homology arms with identical sequences (homologous recombination requires the sequences of the left and right arms to be identical to the genome). These two points restrict the establishment of fixed-point gene knock-in of zebra fish by utilizing a homologous recombination integration method.

At present, two methods for preparing the specific knockout gene zebra fish exist, wherein the first method is to insert LoxP sites at two ends of an exon of a gene respectively in a homologous recombination mode. The method is to insert LoxP site in the way of homologous recombination gene integration, however, the homologous recombination has very low efficiency in the early development of zebra fish, so the success rate of the method is very low. Meanwhile, two LoxP sites need to be inserted in sequence, so that the time is long, and at least more than 6 months are needed.

Another approach is to achieve gene knock-in of specific tissues and cell types by tissue specific promoters driving expression of Cas9 proteins and corresponding sgrnas. However, the uncertainty of the gene sequence caused by the cleavage of the gene of Cas9/sgRNA and the inability to ensure that the target can be cleaved in all cells can cause the disruption of only part of the genes of the cells, and it is difficult to present the phenotype of whole gene knockout, resulting in false negative results.

In response to the above problems, the present inventors skillfully constructed a DNA sequence element containing two LoxP sites into the left arm intron sequence of the knock-in plasmid. In the absence of Cre protein expression, endogenous genes and fluorescent tags with complete structure and function are normally expressed. In the case of expressing Cre protein, the DNA sequence elements of two LoxP sites are inverted by Cre and bind to the previous exon of the gene through splice acceptors, disrupting the structure of the gene, and the mutated gene is also tagged with fluorescent tags of different colors. Therefore, scientific researchers can directly identify whether the gene is knocked out through color under a fluorescence microscope, and the complicated operation that the genotype can be identified only after the PCR amplification and sequencing of a dead living sample in the past is avoided.

In one embodiment of the invention, the probability of a screened foundation that can stabilize the next generation of F1 and be inserted in the forward direction is: about 10% (2/21), 21 microinjected F0 zebra fish were screened, two of which were screened. However, the sequence of the donor plasmid was inserted into the genome of zebra fish by homologous recombination, but no founder was selected, and even if 50 microinjected F0 zebra fish were selected, no founder that could be stably inherited to the next generation was obtained. This suggests that the methods of the present invention may be unexpectedly efficient and stable for site-directed editing (including gene knock-in and/or gene knock-out) of zebra fish genomes.

Due to the randomness of genomic DNA repair after Cas9/sgRNA cleavage. The resulting sequence additions and deletions are random, typically varying from a few base pairs to hundreds of base pairs. Because the technical scheme provided by the invention is carried out in the introns of the genes, the addition and deletion of the basic groups can not influence the target genes to be knocked in and the genes specifically knocked out by tissues. In addition, in consideration of the fact that the knockout efficiency of the scheme is remarkably improved, a plurality of different initial zebra fishes (founder) can be screened and sequenced, and fewer deleted base pairs can be selected as a final experimental target.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Shanghai life science institute of China academy of sciences

<120> method for tissue-specific knockout of zebra fish gene and application thereof

<130> P2019-2050

<160> 12

<170> PatentIn version 3.5

<210> 1

<211> 5136

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> nucleic acid construct I

<400> 1

atgatcttat tttgactaag cgtggctatg aagcagaaag gaaggataat gagttgggta 60

ggttaggtaa gactttctta gacatgagtc aggtcaaaga caacagataa ttccataaaa 120

catatgtatt ttttgtattc tatagaattg tcattaatat tcatgcaaaa gatttcttaa 180

gtgacatttg caaatcactc cagttgtgtc tttctttaca ttgttttcca gttaaattta 240

ggattgattt ctgttattta tttagaataa taattgtata ttaataatga tattgggcag 300

tatattgtac tgtaccctgc tgctggaagg gtatccatat ggggtggttc gcccagggtg 360

ccatttaaac tagaaccatc actgcttcag atggcgtctt taatagttca cattgtatga 420

ttgttacaca agtgaaagag catcgacagg gtaatttgct agcttaccgg tttacgcgta 480

taacttcgta tagcatacat tatacgaagt tatccaagct tcaccatcga cccgaattgc 540

caagcatcac catcgaccca taacttcgta tagtacacat tatacgaagt tatttcgaac 600

gtaatacgac tcactatagg gcgaattgga gctccaccgg tggcggccgc tctagaacta 660

gtggatcctg gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct 720

gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga 780

atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg 840

agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca 900

actagaaggc acagtcgagg ctgatcagcg gtttctggtt ctttccgcct cagaagccat 960

agagcccacc gcatccccag catgcctgct attgtcttcc caatcctccc ccttgctgtc 1020

ctgccccacc ccacccccca gaatagaatg acacctactc agacaatgcg atgcaatttc 1080

ctcattttat taggaaagga cagtgggagt ggcaccttcc agggtcaagg aaggcacggg 1140

ggaggggcaa acaacagatg gctggcaact agaaggcaca gtcgaggctg atcagcgagc 1200

tccaccgcgg tcaattaagt ttgtgcccca gtttgctagg gaggtcgcag tatctggcca 1260

cagccacctc gtgctgctcg acgtaggtct ctttgtcggc ctccttgatt ctttccagtc 1320

tgtggtccac atagtagacg ccgggcatct tgaggttctt agcgggtttc ttggatctgt 1380

atgtggtctt gaagttgcag atcaggtggc ccccgcccac gagcttcagg gccatgtcgc 1440

ttctgccttc caggccgccg tcagcggggt acagcatctc ggtgttggcc tcccagccga 1500

gtgttttctt ctgcatcaca gggccgttgg atgggaagtt cacccctctg atcttgacgt 1560

tgtagatgag gcagccgtcc tggaggctgg tgtcctgggt agcggtcagc acgcccccgt 1620

cttcgtatgt ggtgactctc tcccatgtga agccctcagg gaaggactgc ttaaagaagt 1680

cggggatgcc ctgggtgtgg ttgatgaagg ttctgctgcc gtacatgaag ctggtagcca 1740

ggatgtcgaa ggcgaagggg agagggccgc cctcgaccac cttgattctc atggtctggg 1800

tgccctcgta gggcttgcct tcgccctcgg atgtgcactt gaagtggtgg ttgttcacgg 1860

tgccctccat gtacagcttc atgtgcatgt tctccttaat cagctcttcg cccttagaca 1920

cgacgtcagg tccagggttc tcctccacgt ctccagcctg cttcagcagg ctgaagttag 1980

tagctcttct cttcttccga ccgcgaagag tttgtcgatc gactgaaaaa aaaaagggaa 2040

gagagagaca cgtcagaaac acacacacac tccggattag tgagatctga ataggaactt 2100

cataacttcg tataatgtat gctatacgaa gttatccaag catcaccatc gaccctctag 2160

tccagaactc accatcgacc cataacttcg tataatgtgt actatacgaa gttatactag 2220

tattatgtac ctgactgatc gatttgcctt tgatttctgg catttgtcgg gaatttctca 2280

aaacctgttg tcgagtcaaa atctgggcta aaatcataca gtctgaactc ggctttaggg 2340

gttaataata ttgaccttaa aatggtttta aaagaattaa aaactgcttt tattctagct 2400

gacataaaac aaataagact ttctccagaa gaaaaaaata ttttaggaat tacagtaaaa 2460

aatgtcttgc tctgttaaac atcatttggg aaatatttga acaaaggtat caaaattcac 2520

aggaggtgtg tgtatttaaa gattcactag tatgctcatt tgaataattc tcaatatttt 2580

ttgtcaggat atttcgacgc tcattctctg gccatggact tcttgagcat cggcttccgg 2640

gagtgtctga ctgaagtggc caggtatttg agctctgtgg aaggcctgga ctccagcgac 2700

cctctccgtg tccgtctggt ttctcacctc agcagctgtg cctcgcagag ggaagcagcc 2760

gccatgacca catccatagc ccatcaccag caggcccttc acccgcacca ctgggctgcc 2820

gctttgcatc ccattcctgc tgcgttcctg cagcagagcg gacttccctc ctcagagagc 2880

tcctccggca ggctgtctga ggctcctcaa agaggtgcag cccttttctc ccatagtgac 2940

tcggcactca gagcgccctc tactggaagt gtggctcctt gcgtgccacc gctgtccact 3000

tctctgcttt cgttatcagc gaccgttcat gcagcagctg ctgcagctgc agctcaaacc 3060

ttccctctat catttcccgc tggattccca ctcttcagcc ccagcgttac agcatcttca 3120

gtggcttctt ccaccgtgag ctcttccgtt tccacatcca ccacatccca acagagcagc 3180

gggagcaaca gtaaaccata ccgaccgtgg ggaactgaag tgggagcgtt ttcgggaggt 3240

ggatccggag ctactaattt ctccttgctt aagcaagctg gtgatgttga agaaaatcct 3300

ggtcctatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag 3360

ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc 3420

acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg 3480

cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac 3540

atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc 3600

atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac 3660

accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg 3720

gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag 3780

aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag 3840

ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac 3900

aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac 3960

atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac 4020

aagaccggtt aaatgttgga tttaaatgtt ggacgtcttc catgctttgt acataaagga 4080

aagcagcggc tattgtgcct gcttcggtca gcagcatggg cttttgtctt cctctacact 4140

tgtgcacata tgcagcgtca aacttaagcc aacattctgg gaagaaaaga aagagttttt 4200

acacgtcgca ctgtgttgga aaccgtaaag gaagtttgtt tctgttttaa cagtgcctgc 4260

ataaacactg ctaacatgct gcatttgaga tgtatgcttt gatatcatct gacttccaca 4320

aacacccaac agcagcttta gagtgaacag cttgttctga aacaaaccaa agttttgcag 4380

ataatcacta aagtgaggtg tttgtttttt tatctctgat ttaacaatcc agtttgtaaa 4440

tctgtacatg tgtaagattg taactagagt ttatattgaa attagttcat tggtatgatg 4500

cacttcaatc actactgttt gtttgggggg agacaggatc ttctccgatt tatacaatag 4560

gcctactgaa gttgtttttt taaaataaca ttcactaata ctcatgtgag atttttctac 4620

tactgtaact gtgttaataa ccaccctctg taagatgtaa ccttttccta tgcaaaaaaa 4680

caaatgtccc tcaagaacga actgagtgtg ttttgttttc attctgacac acgctaataa 4740

aaccatcctt ccactagcct tcaccacaac acatcgtgga atgttatgag agaaagtaat 4800

tgttttccca aagcattatt tgagttcttg aaatcgtatg gtagggaaca aatgtttgtg 4860

ctctttaatg tgtttttcta ataatgcaaa atatgcagat gaagtcaaac aaacagctgc 4920

aattgtaacc gccacttcaa cagttataaa tctgtcgaca aactttaaag aaagctacaa 4980

acacatttaa tgaataaaag gtcatcattc ttacatgatc agcagcaaat cggtttactt 5040

tcattgaaaa aagtcaataa tttcttctaa agctaaaata actttttagc tgtgtgtgaa 5100

gagctgtact gtgtgacggt gcctgctaaa acccta 5136

<210> 2

<211> 19

<212> DNA

<213> zebra fish (Danio rerio)

<400> 2

ggaaggataa tggttgggt 19

<210> 3

<211> 717

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> EGFP sequence

<400> 3

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

<210> 4

<211> 34

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> wild-type loxP site

<400> 4

ataacttcgt ataatgtatg ctatacgaag ttat 34

<210> 5

<211> 34

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> mutant lox5171 sequence

<400> 5

ataacttcgt ataatgtgta ctatacgaag ttat 34

<210> 6

<211> 57

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> P2A sequence

<400> 6

aggtccaggg ttctcctcca cgtctccagc ctgcttcagc aggctgaagt tagtagc 57

<210> 7

<211> 225

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> BGHpA Signal sequence

<400> 7

ccatagagcc caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc 60

tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa 120

tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca 180

cgggggaggg gcaaacaaca gatggctggc aactagaagg cacag 225

<210> 8

<211> 236

<212> PRT

<213> synthetic sequence (Artificial sequence)

<220>

<223> TagRFP sequence

<400> 8

Val Ser Lys Gly Glu Glu Leu Ile Lys Glu Asn Met His Met Lys Leu

1 5 10 15

Tyr Met Glu Gly Thr Val Asn Asn His His Phe Lys Cys Thr Ser Glu

20 25 30

Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val

35 40 45

Val Glu Gly Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser

50 55 60

Phe Met Tyr Gly Ser Arg Thr Phe Ile Asn His Thr Gln Gly Ile Pro

65 70 75 80

Asp Phe Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val

85 90 95

Thr Thr Tyr Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser

100 105 110

Leu Gln Asp Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn

115 120 125

Phe Pro Ser Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu

130 135 140

Ala Asn Thr Glu Met Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg

145 150 155 160

Ser Asp Met Ala Leu Lys Leu Val Gly Gly Gly His Leu Ile Cys Asn

165 170 175

Phe Lys Thr Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met

180 185 190

Pro Gly Val Tyr Tyr Val Asp His Arg Leu Glu Arg Ile Lys Glu Ala

195 200 205

Asp Lys Glu Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr

210 215 220

Cys Asp Leu Pro Ser Lys Leu Gly His Lys Leu Asn

225 230 235

<210> 9

<211> 7801

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> hey2zCKOIS donor plasmid

<400> 9

tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga 60

caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct taactatgcg 120

gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc 180

gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa ctgttgggaa 240

gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca 300

aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc 360

agtgaattcg agctcggtac ccactcgtcg acaaaactag ggtttatatg atgatgatct 420

tattttgact aagcgtggct atgaagcaga aaggaaggat aatgagttgg gtaggttagg 480

taagactttc ttagacatga gtcaggtcaa agacaacaga taattccata aaacatatgt 540

attttttgta ttctatagaa ttgtcattaa tattcatgca aaagatttct taagtgacat 600

ttgcaaatca ctccagttgt gtctttcttt acattgtttt ccagttaaat ttaggattga 660

tttctgttat ttatttagaa taataattgt atattaataa tgatattggg cagtatattg 720

tactgtaccc tgctgctgga agggtatcca tatggggtgg ttcgcccagg gtgccattta 780

aactagaacc atcactgctt cagatggcgt ctttaatagt tcacattgta tgattgttac 840

acaagtgaaa gagcatcgac agggtaattt gctagcttac cggtttacgc gtataacttc 900

gtatagcata cattatacga agttatccaa gcttcaccat cgacccgaat tgccaagcat 960

caccatcgac ccataacttc gtatagtaca cattatacga agttatttcg aacgtaatac 1020

gactcactat agggcgaatt ggagctccac cggtggcggc cgctctagaa ctagtggatc 1080

ctggttcttt ccgcctcaga agccatagag cccaccgcat ccccagcatg cctgctattg 1140

tcttcccaat cctccccctt gctgtcctgc cccaccccac cccccagaat agaatgacac 1200

ctactcagac aatgcgatgc aatttcctca ttttattagg aaaggacagt gggagtggca 1260

ccttccaggg tcaaggaagg cacgggggag gggcaaacaa cagatggctg gcaactagaa 1320

ggcacagtcg aggctgatca gcggtttctg gttctttccg cctcagaagc catagagccc 1380

accgcatccc cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc 1440

accccacccc ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt 1500

tattaggaaa ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg 1560

caaacaacag atggctggca actagaaggc acagtcgagg ctgatcagcg agctccaccg 1620

cggtcaatta agtttgtgcc ccagtttgct agggaggtcg cagtatctgg ccacagccac 1680

ctcgtgctgc tcgacgtagg tctctttgtc ggcctccttg attctttcca gtctgtggtc 1740

cacatagtag acgccgggca tcttgaggtt cttagcgggt ttcttggatc tgtatgtggt 1800

cttgaagttg cagatcaggt ggcccccgcc cacgagcttc agggccatgt cgcttctgcc 1860

ttccaggccg ccgtcagcgg ggtacagcat ctcggtgttg gcctcccagc cgagtgtttt 1920

cttctgcatc acagggccgt tggatgggaa gttcacccct ctgatcttga cgttgtagat 1980

gaggcagccg tcctggaggc tggtgtcctg ggtagcggtc agcacgcccc cgtcttcgta 2040

tgtggtgact ctctcccatg tgaagccctc agggaaggac tgcttaaaga agtcggggat 2100

gccctgggtg tggttgatga aggttctgct gccgtacatg aagctggtag ccaggatgtc 2160

gaaggcgaag gggagagggc cgccctcgac caccttgatt ctcatggtct gggtgccctc 2220

gtagggcttg ccttcgccct cggatgtgca cttgaagtgg tggttgttca cggtgccctc 2280

catgtacagc ttcatgtgca tgttctcctt aatcagctct tcgcccttag acacgacgtc 2340

aggtccaggg ttctcctcca cgtctccagc ctgcttcagc aggctgaagt tagtagctct 2400

tctcttcttc cgaccgcgaa gagtttgtcg atcgactgaa aaaaaaaagg gaagagagag 2460

acacgtcaga aacacacaca cactccggat tagtgagatc tgaataggaa cttcataact 2520

tcgtataatg tatgctatac gaagttatcc aagcatcacc atcgaccctc tagtccagaa 2580

ctcaccatcg acccataact tcgtataatg tgtactatac gaagttatac tagtattatg 2640

tacctgactg atcgatttgc ctttgatttc tggcatttgt cgggaatttc tcaaaacctg 2700

ttgtcgagtc aaaatctggg ctaaaatcat acagtctgaa ctcggcttta ggggttaata 2760

atattgacct taaaatggtt ttaaaagaat taaaaactgc ttttattcta gctgacataa 2820

aacaaataag actttctcca gaagaaaaaa atattttagg aattacagta aaaaatgtct 2880

tgctctgtta aacatcattt gggaaatatt tgaacaaagg tatcaaaatt cacaggaggt 2940

gtgtgtattt aaagattcac tagtatgctc atttgaataa ttctcaatat tttttgtcag 3000

gatatttcga cgctcattct ctggccatgg acttcttgag catcggcttc cgggagtgtc 3060

tgactgaagt ggccaggtat ttgagctctg tggaaggcct ggactccagc gaccctctcc 3120

gtgtccgtct ggtttctcac ctcagcagct gtgcctcgca gagggaagca gccgccatga 3180

ccacatccat agcccatcac cagcaggccc ttcacccgca ccactgggct gccgctttgc 3240

atcccattcc tgctgcgttc ctgcagcaga gcggacttcc ctcctcagag agctcctccg 3300

gcaggctgtc tgaggctcct caaagaggtg cagccctttt ctcccatagt gactcggcac 3360

tcagagcgcc ctctactgga agtgtggctc cttgcgtgcc accgctgtcc acttctctgc 3420

tttcgttatc agcgaccgtt catgcagcag ctgctgcagc tgcagctcaa accttccctc 3480

tatcatttcc cgctggattc ccactcttca gccccagcgt tacagcatct tcagtggctt 3540

cttccaccgt gagctcttcc gtttccacat ccaccacatc ccaacagagc agcgggagca 3600

acagtaaacc ataccgaccg tggggaactg aagtgggagc gttttcggga ggtggatccg 3660

gagctactaa tttctccttg cttaagcaag ctggtgatgt tgaagaaaat cctggtccta 3720

tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg 3780

gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg 3840

gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc 3900

tcgtgaccac cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc 3960

agcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct 4020

tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg 4080

tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca 4140

agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg 4200

gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg 4260

accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact 4320

acctgagcac ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc 4380

tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagaccg 4440

gttaaatgtt ggatttaaat gttggacgtc ttccatgctt tgtacataaa ggaaagcagc 4500

ggctattgtg cctgcttcgg tcagcagcat gggcttttgt cttcctctac acttgtgcac 4560

atatgcagcg tcaaacttaa gccaacattc tgggaagaaa agaaagagtt tttacacgtc 4620

gcactgtgtt ggaaaccgta aaggaagttt gtttctgttt taacagtgcc tgcataaaca 4680

ctgctaacat gctgcatttg agatgtatgc tttgatatca tctgacttcc acaaacaccc 4740

aacagcagct ttagagtgaa cagcttgttc tgaaacaaac caaagttttg cagataatca 4800

ctaaagtgag gtgtttgttt ttttatctct gatttaacaa tccagtttgt aaatctgtac 4860

atgtgtaaga ttgtaactag agtttatatt gaaattagtt cattggtatg atgcacttca 4920

atcactactg tttgtttggg gggagacagg atcttctccg atttatacaa taggcctact 4980

gaagttgttt ttttaaaata acattcacta atactcatgt gagatttttc tactactgta 5040

actgtgttaa taaccaccct ctgtaagatg taaccttttc ctatgcaaaa aaacaaatgt 5100

ccctcaagaa cgaactgagt gtgttttgtt ttcattctga cacacgctaa taaaaccatc 5160

cttccactag ccttcaccac aacacatcgt ggaatgttat gagagaaagt aattgttttc 5220

ccaaagcatt atttgagttc ttgaaatcgt atggtaggga acaaatgttt gtgctcttta 5280

atgtgttttt ctaataatgc aaaatatgca gatgaagtca aacaaacagc tgcaattgta 5340

accgccactt caacagttat aaatctgtcg acaaacttta aagaaagcta caaacacatt 5400

taatgaataa aaggtcatca ttcttacatg atcagcagca aatcggttta ctttcattga 5460

aaaaagtcaa taatttcttc taaagctaaa ataacttttt agctgtgtgt gaagagctgt 5520

actgtgtgac ggtgcctgct aaaaccctac tgcaggcatg caagcttggc gtaatcatgg 5580

tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 5640

ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 5700

ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 5760

ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact 5820

gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 5880

atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 5940

caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 6000

cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 6060

taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 6120

ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 6180

tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 6240

gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 6300

ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 6360

aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 6420

agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 6480

agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 6540

cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 6600

gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 6660

atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 6720

gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 6780

tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 6840

gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 6900

ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 6960

actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 7020

ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 7080

tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 7140

cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 7200

ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 7260

ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 7320

tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 7380

agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 7440

atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 7500

gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 7560

aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 7620

tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 7680

aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 7740

gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 7800

c 7801

<210> 10

<211> 4237

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> zCas9 mRNA

<400> 10

ggatcacgac atcgactaca aagacgacga tgataagatg gcccctaaga aaaagagaaa 60

ggtcggaatt cacggagttc ccgctgcaga taaaaagtac agcattggac tggacatcgg 120

aacaaatagc gtgggctggg ctgtgattac tgacgaatat aaggtgccta gcaaaaagtt 180

taaagtgctg ggaaacaccg acagacacag catcaaaaaa aacctgatcg gcgctctgct 240

gtttgatagc ggtgaaactg ccgaggctac tagactgaag agaactgcta gaagaagata 300

taccagaaga aagaatagaa tttgttacct gcaagaaatc tttagcaatg agatggcaaa 360

ggttgacgat agcttctttc atagactgga ggagagcttc ctggtcgagg aggacaagaa 420

gcacgagaga caccccatct tcggaaatat cgtggacgag gtggcatacc atgaaaagta 480

tcctaccatt taccacctga gaaaaaagct ggtggacagc acagacaagg ccgatctgag 540

actgatctac ctggcactgg cccacatgat caaatttaga ggccatttcc tgattgaagg 600

agacctgaac cccgataaca gcgatgttga taaactgttc atccaactgg ttcagaccta 660

taaccaactg tttgaggaga accctattaa cgccagcgga gtggatgcaa aggccatcct 720

gagcgctaga ctgagcaaaa gcagaagact ggaaaatctg atcgcccagc tgcccggcga 780

aaaaaagaat ggactgttcg gcaatctgat tgcactgagc ctgggactga cacctaactt 840

caagagcaat ttcgatctgg ctgaggacgc caaactgcag ctgagcaaag acacatatga 900

tgacgacctg gataacctgc tggcacaaat tggtgaccaa tacgctgacc tgttcctggc 960

tgctaagaat ctgagcgatg ccattctgct gagcgacatc ctgagagtga acacagagat 1020

taccaaggca cccctgagcg caagcatgat taagagatac gacgagcacc accaagatct 1080

gaccctgctg aaggccctgg tcagacaaca actgccagag aagtataaag aaattttctt 1140

tgaccaaagc aagaacggtt acgctggcta cattgacggc ggtgcaagcc aagaggagtt 1200

ctataagttc attaagccaa tcctggagaa aatggatgga actgaggagc tgctggttaa 1260

gctgaataga gaggatctgc tgagaaaaca aagaacattc gacaacggta gcatcccaca 1320

ccagattcat ctgggtgagc tgcacgcaat tctgagaaga caggaagact tttatccatt 1380

cctgaaggac aacagagaaa agatcgagaa gattctgaca tttagaatcc cctactacgt 1440

gggacctctg gctagaggca atagcagatt cgcatggatg actagaaaga gcgaggagac 1500

aattacccct tggaactttg aagaagtggt ggataaggga gcaagcgccc aaagcttcat 1560

tgagagaatg acaaacttcg ataagaacct gcctaacgag aaggttctgc ccaagcatag 1620

cctgctgtat gaatatttca cagtgtacaa cgagctgaca aaggtcaagt acgtcacaga 1680

gggcatgaga aagcccgcct ttctgagcgg agaacaaaag aaggctattg ttgacctgct 1740

gttcaagacc aacagaaaag ttacagttaa acagctgaaa gaggactact tcaaaaagat 1800

tgaatgtttt gacagcgtgg aaatcagcgg cgttgaggac agatttaacg ctagcctggg 1860

cacctaccac gatctgctga aaatcatcaa agataaggac tttctggaca acgaagaaaa 1920

cgaggacatt ctggaagaca ttgtgctgac actgactctg ttcgaagata gagaaatgat 1980

cgaggaaaga ctgaaaactt atgcacatct gttcgacgac aaagtgatga agcaactgaa 2040

gagaagaaga tacactggat ggggcagact gagcagaaag ctgatcaacg gaatcagaga 2100

caagcaaagc ggaaaaacta ttctggattt tctgaaaagc gacggtttcg ccaatagaaa 2160

cttcatgcaa ctgattcacg atgacagcct gactttcaag gaggatattc aaaaggcaca 2220

ggtgagcggc cagggcgata gcctgcacga acacatcgca aatctggccg gtagccctgc 2280

cattaagaag ggcatcctgc agacagtgaa ggttgttgat gaactggtca aggtgatggg 2340

tagacacaag cccgagaata ttgtgatcga gatggctaga gagaaccaaa caacacaaaa 2400

gggacagaag aatagcagag aaagaatgaa aagaattgag gagggaatca aggagctggg 2460

tagccagatc ctgaaagaac accctgtcga gaatacacaa ctgcaaaacg aaaagctgta 2520

cctgtactac ctgcaaaatg gcagagacat gtacgtggac caagagctgg atattaacag 2580

actgagcgac tacgatgtcg accacatcgt gcctcaaagc ttcctgaagg atgacagcat 2640

cgacaataaa gtgctgacta gaagcgacaa gaacagagga aaaagcgaca acgtgcccag 2700

cgaggaagtg gttaaaaaga tgaagaacta ctggagacag ctgctgaatg ccaagctgat 2760

cacacaaaga aaattcgaca acctgaccaa agccgagaga ggaggtctga gcgaactgga 2820

caaggctgga ttcattaaga gacaactggt tgaaaccaga cagattacaa agcacgtggc 2880

tcaaatcctg gacagcagaa tgaataccaa atatgacgag aacgacaaac tgattagaga 2940

ggtgaaggtt attactctga agagcaaact ggtcagcgac ttcagaaagg acttccaatt 3000

ctacaaggtg agagagatca acaattacca ccacgcacac gacgcttacc tgaacgctgt 3060

ggtgggcaca gctctgatca aaaagtatcc aaaactggaa agcgagtttg tgtacggtga 3120

ctataaagtt tatgatgtga gaaaaatgat cgctaagagc gagcaggaga tcggaaaggc 3180

tacagccaag tatttctttt acagcaacat tatgaacttt ttcaagactg aaatcaccct 3240

ggcaaacggt gagatcagaa aaagaccact gatcgaaaca aatggcgaga caggcgagat 3300

cgtgtgggat aagggaagag acttcgctac cgttagaaag gttctgagca tgccacaggt 3360

taacattgtg aagaaaactg aggtgcagac aggaggtttc agcaaggaga gcatcctgcc 3420

taagagaaac agcgataagc tgattgcaag aaaaaaggat tgggacccta agaagtacgg 3480

cggttttgac agccctactg tggcttacag cgtgctggtg gtggctaaag tggagaaggg 3540

caaaagcaag aagctgaaaa gcgtgaagga actgctggga attacaatca tggagagaag 3600

cagcttcgag aagaacccaa tcgacttcct ggaggctaag ggatacaagg aagttaagaa 3660

ggacctgatc atcaagctgc ccaagtacag cctgttcgag ctggaaaatg gtagaaagag 3720

aatgctggct agcgctggtg agctgcagaa gggaaatgaa ctggcactgc ctagcaagta 3780

cgttaacttt ctgtatctgg caagccatta cgagaaactg aaaggaagcc ccgaggacaa 3840

tgagcagaaa caactgttcg tggaacagca caaacactat ctggacgaga ttatcgagca 3900

gatcagcgaa tttagcaaaa gagtgatcct ggctgatgct aacctggata aagtcctgag 3960

cgcttacaac aaacatagag ataagcctat cagagagcag gccgaaaaca tcatccacct 4020

gttcacactg acaaacctgg gcgctcctgc cgctttcaag tactttgata ccactattga 4080

tagaaagaga tatactagca ccaaagaggt gctggacgcc accctgattc accagagcat 4140

taccggactg tacgaaacta gaatcgacct gagccaactg ggaggagaca agagacccgc 4200

tgcaactaaa aaggcaggtc aggccaaaaa gaagaaa 4237

<210> 11

<211> 108

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> hey2 sgRNA

<400> 11

ggaaggataa tggttgggtg ttttagagct agaaatagca agttaaaata aggctagtcc 60

gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttaaagct 108

<210> 12

<211> 997

<212> DNA

<213> synthetic sequence (Artificial sequence)

<220>

<223> Cre recombinase

<400> 12

gcctgcatta ccggtcgatg caacgagtga tgaggttcgc aagaacctga tggacatgtt 60

cagggatcgc caggcgtttt ctgagcatac ctggaaaatg cttctgtccg tttgccggtc 120

gtgggcggca tggtgcaagt tgaataaccg gaaatggttt cccgcagaac ctgaagatgt 180

tcgcgattat cttctatatc ttcaggcgcg cggtctggca gtaaaaacta tccagcaaca 240

tttgggccag ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa gtgacagcaa 300

tgctgtttca ctggttatgc ggcggatccg aaaagaaaac gttgatgccg gtgaacgtgc 360

aaaacaggct ctagcgttcg aacgcactga tttcgaccag gttcgttcac tcatggaaaa 420

tagcgatcgc tgccaggata tacgtaatct ggcatttctg gggattgctt ataacaccct 480

gttacgtata gccgaaattg ccaggatcag ggttaaagat atctcacgta ctgacggtgg 540

gagaatgtta atccatattg gcagaacgaa aacgctggtt agcaccgcag gtgtagagaa 600

ggcacttagc ctgggggtaa ctaaactggt cgagcgatgg atttccgtct ctggtgtagc 660

tgatgatccg aataactacc tgttttgccg ggtcagaaaa aatggtgttg ccgcgccatc 720

tgccaccagc cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa ctcatcgatt 780

gatttacggc gctaaggatg actctggtca gagatacctg gcctggtctg gacacagtgc 840

ccgtgtcgga gccgcgcgag atatggcccg cgctggagtt tcaataccgg agatcatgca 900

agctggtggc tggaccaatg taaatattgt catgaactat atccgtaacc tggatagtga 960

aacaggggca atggtgcgcc tgctggaaga tggcgat 997

Claims

1. A nucleic acid construct I, characterized in that said construct has the structure of formula I from 5 'to 3':

LA-X-RA(I)

in the method, in the process of the invention,

LA, X, RA are elements for constructing the construct, respectively;

each "-" is independently a bond or a nucleotide linking sequence;

LA is the left homologous arm sequence after modification;

x is a first exogenous gene expression cassette;

RA is a right homology arm sequence;

the element LA sequence and the RA sequence enable the construct to generate fixed-point non-homologous recombination with a target segment of a zebra fish chromosome, wherein the target segment is a zebra fish target gene 3 '-end containing an intron, an exon, a terminator and a 3' -UTR segment, and a single guide RNA (sgRNA) target sequence is contained in the intron sequence of the target segment; and is also provided with

The site of LA site-directed recombination is located in the sequence of introns and exons at the 5' end of the target segment up to the terminator, and the LA sequence (5 '. Fwdarw.3 ') comprises the single guide RNA (sgRNA) target sequence and an operably linked nucleic acid construct II;

the nucleic acid construct II has a structure of formula II from 5 'to 3':

L5-L5'-Y-L3-L3'(II)

In the method, in the process of the invention,

l5, L5', X, L, L3' are elements for constructing the construct, respectively;

each "-" is independently a bond or a nucleotide linking sequence;

l5 is a 5' first site-specific recombination sequence;

l5 'is a 5' second site-specific recombination sequence;

y is an inverted second exogenous gene expression cassette;

l3 is a 3' first site-specific recombination sequence;

l3 'is a 3' second site-specific recombination sequence;

the site-specific recombination sequence is a wild type Loxp or a mutant Loxp;

the length of the homologous arm sequence is 200-5000bp.

2. The nucleic acid construct I of claim 1, wherein the first exogenous gene expression cassette X comprises a GSG-P2A self-cleaving sequence and an EGFP sequence.

3. The nucleic acid construct I of claim 2 wherein the EGFP sequence is set forth in SEQ ID NO. 3.

4. The nucleic acid construct I of claim 1, wherein the RA right homology arm sequence starts at the stop codon of the target gene and covers the 3 'gene spacer of the entire 3' utr sequence of the target gene.

5. The nucleic acid construct I of claim 1, wherein the nucleic acid construct I has a nucleic acid sequence as set forth in SEQ ID NO. 1, wherein the nucleic acid sequence of the LA left homology arm sequence is positions 1-3309 of the nucleic acid sequence set forth in SEQ ID NO. 1, the nucleic acid sequence of the first foreign gene expression cassette X is positions 3310-4098 of the nucleic acid sequence set forth in SEQ ID NO. 1, and the nucleic acid sequence of the RA right homology arm is positions 4099-5205 of the nucleic acid sequence set forth in SEQ ID NO. 1.

6. The nucleic acid construct I of claim 1, wherein the first site-specific recombination sequence is a wild-type loxP site.

7. The nucleic acid construct I of claim 1, wherein the second site-specific recombination sequence is a mutant lox5171 site.

8. The nucleic acid construct I of claim 1, wherein the target segment of the nucleic acid construct I is exon 5E 5 of the zebra fish hey2 gene and an intron sequence comprising the sgRNA target sequence.

9. The nucleic acid construct I of claim 1, wherein the sgRNA target sequence targets an intron sequence of the hey gene.

10. The nucleic acid construct I of claim 9, wherein the intron sequence, hey sgRNA target sequence, is GGAAGGATAATGGTTGGGT.

11. The nucleic acid construct I of claim 1, wherein the nucleic acid construct II has the nucleic acid sequence set forth in SEQ ID NO. 1 at positions 468-2271.

12. A vector comprising the construct of claim 1.

13. The vector of claim 12, wherein the vector is a hey zCKOIS donor plasmid having the sequence set forth in SEQ ID No. 9.

14. A reagent, wherein the reagent comprises: (a) Cas9 mRNA, (b) target gene sgRNA, and (c) the nucleic acid construct of claim 1 and/or the vector of claim 12, wherein the single guide RNA target sequence contained in the nucleic acid construct and/or the vector corresponds to (b) target gene sgRNA sequence.

15. A host cell comprising the construct of claim 1, or having one or more constructs of claim 1 integrated into its genome.

16. A method for preparing a transgenic cell in vitro comprising the steps of:

(i) Transfecting a cell with the construct of claim 1 and/or the vector of claim 12 such that the construct undergoes site-directed non-homologous recombination with a chromosome in the cell, thereby producing a transgenic cell.

17. A method for preparing a transgenic cell in vitro comprising the steps of:

(i) Transfecting a cell with the construct of claim 1 and/or the vector of claim 12 in the presence of Cre recombinase such that the construct undergoes site-directed non-homologous recombination with a chromosome in the cell, thereby producing a transgenic cell.

18. A method of making a transgenic animal comprising the steps of:

(i) Transfecting a cell with the construct of claim 1 and/or the vector of claim 12 such that the construct undergoes site-directed recombination with a chromosome in the cell, thereby producing a transgenic cell, and wherein the site of site-directed cleavage is located at a chromosomal target segment of a zebra fish; and

19. A method of making a tissue-specific transgenic animal comprising the steps of:

(a) Preparing a transgenic animal F1 with a genome stably inserted construct according to claim 1 according to the method of claim 18;