CN109666702A - A kind of CRISPR/Cas9 carrier can be used for insect genes editor - Google Patents

A kind of CRISPR/Cas9 carrier can be used for insect genes editor Download PDF

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CN109666702A
CN109666702A CN201910156588.2A CN201910156588A CN109666702A CN 109666702 A CN109666702 A CN 109666702A CN 201910156588 A CN201910156588 A CN 201910156588A CN 109666702 A CN109666702 A CN 109666702A
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sgrna
gene
dna
crispr
cas9
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唐雪明
李学文
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Silicon Yihe Technology (shanghai) Co Ltd
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Silicon Yihe Technology (shanghai) Co Ltd
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • A01K2217/07—Animals genetically altered by homologous recombination
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract

The present invention provides a kind of CRISPR/Cas9 carriers that can be used for insect genes editor.Specifically, the present invention provides a kind of for editing the reagent of insect cell gene, and the reagent includes the sgRNA for targeting ABC transporter gene or the expression vector for expressing the sgRNA.Of the invention avoids using chemical pesticide, protects environment, has ensured food safety, and the pest generated is not likely to produce drug resistance.

Description

A kind of CRISPR/Cas9 carrier can be used for insect genes editor
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of CRISPR/Cas9 load that can be used for insect genes editor Body.
Background technique
CRISPR (clustered regularly interspaced short palindromic repeats) technology It is the new and effective gene editing technology that developed recently gets up, from the research to bacterial immune mechanism.In phage-infect During bacterium, bacterium is integrated into the Partial Fragment of bacteriophage nucleic acid in autogene group, and evolves Cas (CRISPR- Associated) nuclease.When bacteriophage infects again, Cas nuclease is tied under the guidance of these segments (referred to as gRNA) It closes the corresponding site of bacteriophage nucleic acid and cuts nucleic acid, to prevent infecting for bacteriophage.Researcher utilizes this principle, hair A kind of gene editing technology has been opened up, i.e., has guided Cas9 nuclease to target gene in sequence by gRNA, Cas9 cutting is double Chain DNA introduces the mutation such as missing insertion by non-homologous end joining mechanism, to destroy gene function.
CRISPR/Cas9 gene editing technology needs corresponding carrier to realize.Basic process is from streptococcus pyogenes Cas9 gene order is obtained in (Streptococcus pyogenes) and passes through particular expression host codon optimization rear clone To corresponding expression vector, and clone's tiny RNA promoter driving gRNA frame sequence to same expression vector.
CRISPR/Cas9 gene editing technology is to be determined by gRNA to the selection of target gene editing sites, and gRNA Design needs to follow certain rule.First, transcription initiation base is guanine G；Second, intermediate sequence length is 20 alkali Base；Third is necessary for NGG, referred to as PAM sequence with three base of above-mentioned 20 base zone downstreams in target gene, and wherein N, which is represented, appoints One base.
Agricultural pest management is a very important link in agricultural production.For planting industry, China is universal at present Using chemical pesticide, the supply of grain and fruits and vegetables ensure that, but equally to human health, ecology in its production and use process Environment and species diversity cause deep effect, therefore there is an urgent need in the art to develop new agricultural pest management means.
Summary of the invention
It is an object of the invention to provide a kind of CRISPR/Cas9 carriers that can be used for insect genes editor.
In the first aspect of the present invention, provide a kind of for editing the reagent of insect cell gene, the reagent includes:
(i) sgRNA of ABC transporter gene or the expression vector for expressing the sgRNA are targeted；With
The expression cassette of optional (ii) expression Cas9 albumen or expression Cas9 albumen.
In another preferred example, the insect is diamondback moth, striped rice borer.
In another preferred example, the ABC transporter gene is ABCH1.
In another preferred example, the sgRNA includes the nucleotide that identification region sequence is corresponded to containing it.
In another preferred example, the sequence of the sgRNA is that sgRNA identifies region sequence or corresponding overall length sgRNA sequence.
In another preferred example, the sequence of the sgRNA includes sequence selected from the group below: (1) sgRNA identifies region sequence； Or (2) corresponding overall length sgRNA sequence.
In another preferred example, the overall length sgRNA sequence successively identifies region sequence containing sgRNA from 5 ' ends to 3 ' ends With sgRNA Frame sequence.
In another preferred example, the sgRNA identification region sequence has SEQ ID NO.:1 (GTCGCCGCCGGGCGACGAGA) nucleotide sequence shown in.
In another preferred example, the sgRNA Frame sequence has SEQ ID NO.:2 (GTTTTAGAGCTAGAAATAG CAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG C) shown in nucleotide sequence.
In another preferred example, the sgRNA has SEQ ID No.:3 (GTCGCCGCCGGGCGACGAGAGTTTTAG AGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGC ACCGAGTCGGTGC) shown in Nucleotide sequence.
In another preferred example, the sgRNA of the targeting ABC transporter gene is to pass through or repair without chemical group Decorations.
In another preferred example, the ABC transporter gene derives from insect, preferably, deriving from diamondback moth.
In the second aspect of the present invention, a kind of kit is provided, the kit contains:
(1) the first container and the first expression vector in the first container, first expression vector contain First expression cassette, first expression cassette are used to express the sgRNA of the targeting ABC transporter gene；With
Optional (2) second container and the second expression vector in the second container, second expression carry Body contains the second expression cassette, and second expression cassette is used to express the expression cassette of Cas9 albumen.
In another preferred example, the identification region sequence of the sgRNA of the targeting ABC transporter gene has SEQ ID Nucleotide sequence shown in NO.:1.
In another preferred example, first and second expression vector is independent or is connected.
In another preferred example, first and second expression vector is located in identical or different container.
In another preferred example, first and second expression cassette is located on identical or different carrier.
In another preferred example, the carrier is selected from the group: DNA, RNA, plasmid, slow virus carrier, adenovirus vector, Retroviral vector, transposons, other gene transfer systems, or combinations thereof.
In another preferred example, the expression vector includes pAc-sgRNA-Cas9.
In another preferred example, the sequence of the expression vector such as SEQ ID No.:4 (ggaaaaatgatgtgacagtg gaaatgagaagtaggtgcatctgcaaagatttgattcagagttgatgccattcatgattttttttagaagtatcat ttgtgtttttagaatatagaattaattccttgaagacgaaagggcctcgtgatacgcctatttttataggttaatg tcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatt tttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaagg aagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctc acccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatct caacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta tgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgact tggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccat aaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttg cacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagc gtgacaccacgatgcctgcagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttc ccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc tggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggta agccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctga gataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaa cttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagt tttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaat ctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttt ccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccact tcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataa gtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagcattgagaaagcgcca cgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagct tccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtga tgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggc tggtaagagccgcgagcgatccttgaagctgtccctgatggtcgtcatctacctgcctggacagcatggcctgcaa cgcgggcatcccgatgccgccggaagcgagaagaatcataatggggaaggccatccagcctcgcgtcgcgaacgcc agcaagacgtagcccagcgcgtcggccgccatgccggcgataatggcctgcttctcgccgaaacgtttggtggcgg gaccagtgacgaaggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgcgct ccagcgaaagcggtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaag acagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagg gcatcggtcgactgatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacct ccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaa taaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactca tcaatgtatcttatcatgtctggatccctcgagtcaggcaccgggcttgcgggtcatgcaccaggtgcgcggtcct tcgggcacctcgacgtcggcggtgacggtgaagccgagccgctcgtagaaggggaggttgcggggcgcggaggtct ccaggaaggcgggcaccccggcgcgctcggccgcctccactccggggagcacgacggcgctgcccagacccttgcc ctggtggtcgggcgagacgccgacggtggccaggaaccacgcgggctccttgggccggtgcggcgccaggaggcct tccatctgttgctgcgcggccagccgggaaccgctcaactcggccatgcgcgggccgatctcggcgaacaccgccc ccgcttcgacgctctccggcgtggtccagaccgccaccgcggcgccgtcgtccgcgacccacaccttgccgatgtc gagcccgacgcgcgtgaggaagagttcttgcagctcggtgacccgctcgatgtggcggtccgggtcgacggtgtgg cgcgtggcggggtagtcggcgaacgcggcggcgagggtgcgtacggcccgggggacgtcgtcgcgggtggcgaggc gcaccgtgggcttgtactcggtcatgctaccgctgccgctaccgctagcagggccgggattctcctccacgtcacc gcatgttagaagacttcctctgccctcaagcttgcctgatccctttttcttttttgcctggccggcctttttcgtg gccgccggccttttgtcgcctcccagctgagacaggtcgatccgtgtctcgtacaggccggtgatgctctggtgga tcagggtggcgtccagcacctctttggtgctggtgtacctcttccggtcgatggtggtgtcaaagtacttgaaggc ggcaggggctcccagattggtcagggtaaacaggtggatgatattctcggcctgctctctgatgggcttatcccgg tgcttgttgtaggcggacagcactttgtccagattagcgtcggccaggatcactctcttggagaactcgctgatct gctcgatgatctcgtccaggtagtgcttgtgctgttccacaaacagctgtttctgctcattatcctcgggggagcc cttcagcttctcatagtggctggccaggtacaggaagttcacatatttggagggcagggccagttcgtttcccttc tgcagttcgccggcagaggccagcattctcttccggccgttttccagctcgaacagggagtacttaggcagcttga tgatcaggtcctttttcacttctttgtagcccttggcttccagaaagtcgatgggattcttctcgaagctgcttct ttccatgatggtgatccccagcagctctttcacactcttcagtttcttggacttgcccttttccactttggccacc accagcacagaataggccacggtggggctgtcgaagccgccgtacttcttagggtcccagtccttctttctggcga tcagcttatcgctgttcctcttgggcaggatagactctttgctgaagccgcctgtctgcacctcggtctttttcac gatattcacttggggcatgctcagcactttccgcacggtggcaaaatcccggcccttatcccacacgatctccccg gtttcgccgtttgtctcgatcagaggccgcttccggatctcgccgttggccagggtaatctcggtcttgaaaaagt tcatgatgttgctgtagaagaagtacttggcggtagccttgccgatttcctgctcgctcttggcgatcatcttccg cacgtcgtacaccttgtagtcgccgtacacgaactcgctttccagcttagggtactttttgatcagggcggttccc acgacggcgttcaggtaggcgtcgtgggcgtggtggtagttgttgatctcgcgcactttgtaaaactggaaatcct tccggaaatcggacaccagcttggacttcagggtgatcactttcacttcccggatcagcttgtcattctcgtcgta cttagtgttcatccgggagtccaggatctgtgccacgtgctttgtgatctgccgggtttccaccagctgtctcttg atgaagccggccttatccagttcgctcaggccgcctctctcggccttggtcagattgtcgaactttctctgggtaa tcagcttggcgttcagcagctgccgccagtagttcttcatcttcttcacgacctcttcggagggcacgttgtcgct cttgccccggttcttgtcgcttctggtcagcaccttgttgtcgatggagtcgtccttcagaaagctctgaggcacg atatggtccacatcgtagtcggacagccggttgatgtccagttcctggtccacgtacatatcccgcccattctgca ggtagtacaggtacagcttctcgttctgcagctgggtgttttccacggggtgttctttcaggatctggctgcccag ctctttgatgccctcttcgatccgcttcattctctcgcggctgttcttctgtcccttctgggtggtctggttctct ctggccatttcgatcacgatgttctcgggcttgtgccggcccatcactttcacgagctcgtccaccaccttcactg tctgcaggatgcccttcttaatggcggggctgccggccagattggcaatgtgctcgtgcaggctatcgccctggcc ggacacctgggctttctggatgtcctctttaaaggtcaggctgtcgtcgtggatcagctgcatgaagtttctgttg gcgaagccgtcggacttcaggaaatccaggattgtcttgccggactgcttgtcccggatgccgttgatcagcttcc ggctcagcctgccccagccggtgtatctccgccgcttcagctgcttcatcactttgtcgtcgaacaggtgggcata ggttttcagccgttcctcgatcatctctctgtcctcaaacagtgtcagggtcagcacgatatcttccagaatgtcc tcgttttcctcattgtccaggaagtccttgtccttgataattttcagcagatcgtggtatgtgcccagggaggcgt tgaaccgatcttccacgccggagatttccacggagtcgaagcactcgattttcttgaagtagtcctctttcagctg cttcacggtcactttccggttggtcttgaacagcaggtccacgatggcctttttctgctcgccgctcaggaaggcg ggctttctcattccctcggtcacgtatttcactttggtcagctcgttatacacggtgaagtactcgtacagcaggc tgtgcttgggcagcaccttctcgttgggcaggttcttatcgaagttggtcatccgctcgatgaagctctgggcgga agcgcccttgtccaccacttcctcgaagttccagggggtgatggtttcctcgctctttctggtcatccaggcgaat ctgctgtttcccctggccagagggcccacgtagtaggggatgcggaaggtcaggatcttctcgatcttttcccggt tgtccttcaggaatgggtaaaaatcttcctgccgccgcagaatggcgtgcagctctcccaggtggatctggtgggg gatgctgccgttgtcgaaggtccgctgcttccgcagcaggtcctctctgttcagcttcacgagcagttcctcggtg ccgtccatcttttccaggatgggcttgatgaacttgtagaactcttcctggctggctccgccgtcaatgtagccgg cgtagccgttcttgctctggtcgaagaaaatctctttgtacttctcaggcagctgctgccgcacgagagctttcag cagggtcaggtcctggtggtgctcgtcgtatctcttgatcatagaggcgctcaggggggccttggtgatctcggtg ttcactctcaggatgtcgctcagcaggatggcgtcggacaggttcttggcggccagaaacaggtcggcgtactggt cgccgatctgggccagcaggttgtccaggtcgtcgtcgtaggtgtccttgctcagctgcagtttggcatcctcggc caggtcgaagttgctcttgaagttgggggtcaggcccaggctcagggcaatcaggtttccgaacaggccattcttc ttctcgccgggcagctgggcgatcagattttccagccgtctgctcttgctcagtctggcagacaggatggccttgg cgtccacgccgctggcgttgatggggttttcctcgaacagctggttgtaggtctgcaccagctggatgaacagctt gtccacgtcgctgttgtcggggttcaggtcgccctcgatcaggaagtggccccggaacttgatcatgtgggccagg gccagatagatcagccgcaggtcggccttgtcggtgctgtccaccagtttctttctcaggtggtagatggtggggt acttctcgtggtaggccacctcgtccacgatgttgccgaagatggggtgccgctcgtgcttcttatcctcttccac caggaaggactcttccagtctgtggaagaagctgtcgtccaccttggccatctcgttgctgaagatctcttgcaga tagcagatccggttcttccgtctggtgtatcttcttctggcggttctcttcagccgggtggcctcggctgtttcgc cgctgtcgaacagcagggctccgatcaggttcttcttgatgctgtgccggtcggtgttgcccagcaccttgaattt cttgctgggcaccttgtactcgtcggtgatcacggcccagcccacagagttggtgccgatgtccaggccgatgctg tacttcttgtcggctgctgggactccgtggataccgaccttccgcttcttctttggggccatcttatcgtcatcgt ctttgtaatcaatatcatgatccttgtagtctccgtcgtggtccttatagtccatgaattccaccacactggacta gtaggtaccccgatccggggtctctggattagacgactgctggctgatggagcggctttgtgtcgggaggagtatc cacacagcacaagaactcaaacggtagtgatatgaaaactggtcccgcagcccgttttataaagcccgcttagcgc taaaaccttatcagccgcaatgctctccgctctcaagtcgcgttcaaaacttttaccatatatgggtatgaattgt tcgccgattgggttttcatttacggagaatttcctccgcaactgggtgttcgtgcttgtgtgtgtcttgtatatcg ccatgaagagtgtacttccatgggtcctatcagttgcggcatgtgattcaatttgggggttactcatattcgatac ttcagctggttatctttatgctgtgatggtcacggtgcttttacacattttttccccaactactcattgtatgcca aaacatacatatgtacatgtgtttatagtgggtttattagtaatgtccagcgatattaaaacattacagttaaata atattcatattagcttacgtttactgctatctgcacttcaaattggttattcagggacatttgtgcaacacacatc ttctagtaggggcatcactagtcgtagaaaacacacctaacttaagccggttttaacaataacggcgaaacagctg ccagacgagagaaaagtacacagcaagaagcatttctataatgatgccgtcctaaatatgcagcatttttgctcaa attgatcatttcgttgcatcgctcaggtggtatagtatttggctattgaatgtgattgctatcttaatagttttga atagtaatagtgcaaatattcattcataaaaaataggtaatcgattttctcagtgcagtctatgcgtttgcgcaaa caggagacgaagagagagacgcctcacacacaagcacatgcggtctaagctgctctccctctttttctctctctct ctgcttggcggcagttcttccagaggctgtgacgtcaggtaagctatttcgtctcggtgtgctgttagcccgtaac gataagctgtccgattcctaattctctttcaaataatttgttttatttttttttttgtgtgttttgaagtcaaaca gcttcataaacaatattaaatatcgcaattgttattgcaaatgcattcgtatcaggcgccttgatgtttttcggcc aaaacttcgtctgctctttgtcagcgtaattgcgtttgccggtcgtgccgtcttgctcgcacgcgcttgcaaacaa aaagtgaaagagggaaagccactgtgttgttgcttttgttgctatcgttggctgcacacacatggcaataagtaac catattgtagacttatttattttagcgttttcgttgactaatctttgtttaaataattgcctatttcgagccaaaa tgacgcataaaggacacggcaacatgcggtgcgttgagcatgcgtacgagatggcgctagagagagcgagataaaa agaaaaaaacaaccggcgagtggcgagacacgaataatacacacacgcacacatggtcaaaggagtggctccaacg tcggtgacgtcgcgcccagccacagaccccgccacgccccgccccacccttccttccgctgccccgcacccctttc cttccactaccgttttgccgcatttggtattgccatctcgcttttttggctttactttgctttttgaataagccaa gaaactgaaatcaaagcggacagtccgaaaattgaaactattccaaaaaacaacaaccgcacatgcactgcgttcg gcaaaagacgcagaaatcagcctgtggtctggcatcatcttaaaatatcatcagtatcatcattgccattataaat acataagaaaatgagaatacattaaaggtatatacatacaatctttgtcatagtcaacatattttgcaacaaaatt atctatagataacatgtttttgtagaaaattccactaactgattttcgtataaatacaaattattctcattcttca aaatactcgccgacgtcatcgcgaaattcctcagcattcttcgacaaatgaagagagacagcgagtgacacaatct tatcttgccgacgagtctttgaaaatggccccgggtaacggtgaaaaacatatagccaagaatggagcgatcgcca ggcgatggaaattggcttcaaattgctccgaatatggccagttggtcagttggcattggattgtggagagatacta gatcagctcgttctccttctcctccgcattctcctactccgcccgaagacgcttctttgatcgcttctttctcgaa tgtgtacatattcattatgtatttggtgtttggcgacgatcagccagctttaatggccataaaacgtgtcttggtt tgtgggccacataacaggcagcgagatctgttcgacttgcagcctgaaatacggcacgagtaggaaaagccgagtc aaatgccgaatgcagagtctcattacagcacaatcaactcaagaaaaactcgacacttttttaccatttgcactta aatccttttttattcgttatgtatactttttttggtccctaaccaaaacaaaaccaaactctcttagtcgtgcctc tatatttaaaactatcaatttattatagtcaataaatcgaactgtgttttcaacaaacgaacaataggacactttg attctaaaggaaattttgaaaatcttaagcagagggttcttaagaccatttgccaattcttataattctcaactgc tctttcctgatgttgatcatttatataggtatgttttcctcaatacttcggaagagcgagctcttccgttttagag ctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttaga Tct shown in).
In the third aspect of the present invention, a kind of method that gene editing is carried out in insect cell, the method are provided Include:
The reagent described in first aspect present invention handles insect cell, to carry out gene editing.
In another preferred example, the insect cell is the cell of in vitro culture.
In another preferred example, the insect is diamondback moth, striped rice borer.
In the fourth aspect of the present invention, a kind of purposes of reagent as described in the first aspect of the invention, the examination are provided Agent is used to prepare kit, and the kit is used for: (a) knocking out ABC transporter gene；And/or it is (b) thin for editing insect Born of the same parents' gene.
In the fifth aspect of the invention, a kind of sgRNA is provided, the sgRNA targeting ABC transporter gene sgRNA。
In another preferred example, the identification region sequence of the sgRNA has nucleotide sequence shown in SEQ ID NO.:1.
In the sixth aspect of the present invention, a kind of carrier is provided, the carrier includes described in fifth aspect present invention sgRNA。
In the seventh aspect of the present invention, a kind of gene editing system is provided, the gene editing system includes such as this hair SgRNA described in bright 5th aspect.
In another preferred example, the gene editing system is CRISPR/Cas system, it is therefore preferable to CRISPR/Cas9 system System.
In another preferred example, gene inactivation editor, the gene inactivation of acquisition are carried out using the CRISPR/Cas9 system Rate >=70%, preferably >=80%, more preferably >=90%.
In the eighth aspect of the present invention, a kind of disinsection method is provided, which comprises
The reagent described in first aspect present invention handles insect cell, to carry out gene editing.
In another preferred example, the insect cell is the cell of in vitro culture.
In another preferred example, the insect is diamondback moth, striped rice borer.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 shows used carrier structural schematic diagram of the present invention.
Fig. 2 shows pAc-Cas9-ABCH1.exon2-gRNA plasmid order-checking result.
Fig. 3 shows pAc-Cas9-ABCH1.exon13-gRNA plasmid order-checking result.
Fig. 4 shows the working principle and gRNA design rule of CRISPR/Cas9.
Specific embodiment
The present inventor after extensive and in-depth study, develops a kind of suitable for insect cell gene editor's for the first time CRISPR/Cas9 carrier construction method and the plasmid of application its method building.Using the target gene ABCH1 of the method building Edit plasmid can transfection insect cell, using CRISPR working mechanism to genomic DNA carry out accurate edits, to be managed The phenotype thought can be applied to agricultural pest management, to substitute chemical pesticide.The present invention is completed on this basis.
Term
Unless otherwise defined, the meaning of all technical and scientific terms used herein and the common skill of fields of the present invention Art personnel are generally understood identical.All publications and other bibliography addressed herein are all incorporated by reference this Text.
As used herein, described " containing ", " having " or " comprising " include "comprising", " mainly by ... constitute ", " substantially by ... constitute " and " by ... constitute ".
CRISPR/Cas gene editing technology
So-called " CRISPR/Cas technology ", " CRISPR/Cas genome editor ", " CRISPR/Cas genome of the invention Editing technique ", " CRISPR/Cas genome edit methods " generally refer to using CRISPR/Cas system to target DNA sequence into The technology of row modification." CRISPR/Cas technology " also may include the method for carrying out gene expression regulation using similar principles, such as Gene expression regulation technology based on CRISPR/dCas9.
CRISPR (Clustered regularly interspaced short palindromic repeats), entirely Referred to as the short palindrome in regular cluster interval repeats, and is the gene editing tool quickly grown in recent years, is originally found to be bacterium use It is a kind of genetic weapon for tackling attacker to protect themselves against the acquired immune system response phenomenon of virus infection.Afterwards Come, it was discovered by researchers that it can mediate gene by being transformed and developing into accurate and efficient gene editing tool Deletion, insertion, the mutation of group sequence, or even target gene can also be activated or inhibited, these target genes include people, mouse, spot Gene in horse fish, bacterium, drosophila, yeast, nematode and crop plant cells, it is meant that CRISPR/Cas9 is that one kind can be extensive The gene editing tool used.Currently, the CRISPR-Cas9 system application from Streptococcus pyogenes is the widest It is general.Nuclease Cas9 albumen in the system contains there are two nuclease domain, can be respectively cut DNA two it is single-stranded. The working principle of CRISPR/Cas9 system is that Cas9 is combined into compound with crRNA and tracrRNA first, then passes through PAM Sequence combination specific dna sequence forms RNA-DNA composite construction, and then cuts to target DNA double-strand, keeps DNA double chain disconnected It splitting, subsequent body repairs the double-strand break of appearance by the link of starting nonhomologous end or homologous recombination machinery, after And mediate the missing of nucleotide sequence, insertion etc..
SgRNA of the invention
As used herein, " sgRNA ", " guide RNA " or " sgRNA of the present invention " is used interchangeably, and refers both to targeting abc transport The sgRNA of protein gene, the sgRNA have nucleotide sequence shown in SEQ ID No.:1: GTCGCCGCCGGGCGACGAGA。
Cas9 albumen
Cas9 albumen is multifunctional protein, and protein structure includes the cog region (REC) of alpha-helix composition, by HNH structure The nuclease area that domain and RuvC structural domain form and the combined area PAM positioned at the end C-.The two important nuclease domains RuvC and HNH can be cut the DNA complementary strand to gRNA with incomplementarity chain respectively, generate the DNA double chain fracture of flat end.When D10A in RuvC structural domain can lead to the inactivation of RuvC structural domain when mutating, when the H840A in HNH structural domain occurs to dash forward It then can lead to the inactivation of HNH structural domain when change.Single-point mutants can make Cas9 become nickase (nickase), referred to as NCas9 can form single-strand DNA breaks.The identification of Cas9 and target DNA is dependent on tracrRNA:crRNA complex and is located at target The PAM sequence in site downstream.
The CRISPR/Cas system that sgRNA of the invention is mediated
On the basis of sgRNA of the invention, the present invention provides the gene editing system comprising the sgRNA, preferably Ground is CRISPR/Cas system.
As known to those skilled in the art, CRISPR/Cas system can be used for various fields, including but not limited to genome Editor, gene expression regulation and genetic engineering etc..In a particular embodiment, CRISPR/Cas system of the invention is used for Genome editor and gene expression regulation.
In a particular embodiment, the CRISPR/Cas system include but is not limited to CRISPR/Cas9 system, CRISPR/nCas9 system or CRISPR/dCas9 system；Preferably, the CRISPR/Cas system is CRISPR/Cas9 system System.
As described above, the core of CRISPR/Cas system of the invention is Cas9 albumen and sgRNA, the two can be same It can also be in different expression vectors in one expression vector.However, since sgRNA and Cas9 albumen is in different expression vectors Reduce sgRNA expression cassette to be subcloned to the process of Cas9 plasmid, so that operation is more easy, therefore, in preferred embodiment party In formula, by the expression plasmid cotransformation of the expression cassette of sgRNA and Cas9.
Common technical knowledge based on technology contents of the invention and this field, as known to those skilled in the art about Cas9's The various technical essentials of expression, the selection of targeting sequence and DNA conversion system, such as those skilled in the art are referred to Nodvig etc. (Nodvig et al., 2015) expression for carrying out Cas9 and implement DNA conversion.
Herein, term " gene inactivation " refers on the basis of not being related to donor dna, by Cas9 drawing in sgRNA After leading the lower progress double-strand cutting to specific site, DNA sequence dna insertion is introduced by non-homologous end joining system in eucaryote Or base deletion etc. and make a certain gene lose biological function；And term " gene is precisely edited " refers to DNA specific Point carry out accurately, predictable genetic manipulation, such as the knockouts of target DNA fragments, insertion, replacement, point mutation etc. relate to And donor dna and homologous recombination machinery.Therefore, as known to those skilled in the art, the difficulty that gene is precisely edited will be much higher than Gene inactivation.In addition, as known to those skilled in the art, the efficiency of the length of donor DNA segment donor dna for genome editor Also have a significant impact.DNA homologous recombination efficiency can be improved to improve DNA in the homologous arm lengths increase of donor DNA segment donor dna Editorial efficiency, but in most eukaryon wild strain its homologous recombination efficiency it is very low, the only efficiency of a few percent sometimes, mainly It is that non-homologous end joining system plays a role.And for the donor DNA segment donor DNA segment of short homology arm, genome Editorial efficiency is then lower.
However, the genome editing system that sgRNA of the invention is mediated has excellent genome compared with the prior art Edit capability.In specific embodiment example, it is higher than using the gene inactivation rate that genome editing system of the invention obtains 95%, more preferably up to 100%.
Main advantages of the present invention include:
1) present invention inactivates ABCH1 albumen accurately using CRISPR/Cas9 method to be precisely controlled the population of certain pest Quantity avoids and injures other beneficial insects；
2) present method avoids chemical pesticide is used, environment is protected；
3) pest that method of the invention generates is not likely to produce drug resistance.
4) present method avoids chemical pesticide is used, food safety has been ensured.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.
Universal method
1) the gRNA design of specific gene
CRISPR/Cas9 method is the scheme for being directed to genomic DNA editor and developing, therefore gRNA design must be pair with DNA As design process is shown in network addresshttp://crispr.mit.edu/.The production of gRNA carrier is annealed by positive sequence and reverse sequence It forms.Designed oligonucleotides carries out DNA synthesis.
2) denaturation and renaturation.The above oligonucleotides after composition, is dissolved in water into 100uM.It is positive for every gRNA Sequence and reverse sequence respectively add 10uL, and add ddH2O 2OuL is heated to 98 DEG C, cooled to room temperature after mixing.
3) phosphorylation Oligo.For every gRNA, step 2) annealed product 1uL, T4 polynueleotide kinase (NEB) is taken 1uL, T4 DNA ligase buffer 1uL, 37 DEG C of incubation 30min after mixing, after the completion plus ddH2O 9OuL to 100uL.
4) carrier digestion.Configure reaction system: pAc-sgRNA-Cas9 carrier 2ug, BspQI (NEB, 10U/uL) restriction endonuclease 1uL, 10 × NEBuffer inscribe enzyme buffer liquid 5uL add ddH2O to total volume 50uL.50 DEG C are incubated for 2 hours, then 80 DEG C 20 minutes progress heat inactivations are incubated for, glue recycles carrier segments.
5) it connects.For every gRNA: in 0.2mL centrifuge tube add above-mentioned steps 3) described in Phosphorylated products 2uL, Add it is above-mentioned 4) in digestion support products 50ng, T4 DNA ligase 1uL and T4 DNA ligase buffer to total volume 10uL is incubated for 2 hours for 18 DEG C after mixing.
6) it converts.For every gRNA, above-mentioned steps 5) connection product conversion DH5 α competent cell, it is coated on containing ammonia Parasiticin (Ampr) LB resistant panel on, it is 6 small that 37 DEG C of LB culture medium cultures are added in 37 DEG C of overnight incubations, picking individual colonies When, extract recombinant plasmid.
7) it identifies.For every gRNA, identification recombination is sequenced with U6Fwd primer (CGAGATCTGTTCGACTTGCAGCC) Plasmid.Sequencing result must quality reach a standard, as a result reliably.
Embodiment 1.
The present embodiment is specifically described by taking agricultural pests diamondback moth as an example.
1) ABCH1 gene gRNA is designed
To be mutated ABCH1 gene, its protein structure is destroyed, gRNA can only be found in each exon.Above-mentioned ABCH1 base Because the gene of containing 13 exons and long introne, 13 exon sequences are shown in SEQ ID NO.11-25 (table 2). The present invention selects the 2nd to carry out gRNA design with the 13rd exon, and sequence is SEQ ID NO.5-8 (table 1), the oligonucleotides It is synthesized by Sheng Gong biotech firm.
2) denaturation and renaturation.The above oligonucleotides after composition, is dissolved in water into 100uM.For exon2, exon2F Respectively add 10uL with exon2R, and adds ddH2O 2OuL is heated to 98 DEG C, cooled to room temperature after mixing.For exon13, Exon13F and exon13R respectively adds 10uL, and adds ddH2O2OuL is heated to 98 DEG C, cooled to room temperature after mixing.
3) phosphorylation Oligo.Exon2 annealed product 1uL, T4 polynueleotide kinase (NEB) 1uL, T4 DNA ligase Buffer 1uL, 37 DEG C of incubation 30min after mixing, after the completion plus ddH2O 9OuL to 100uL.Exon13 annealed product 1uL, T4 Polynueleotide kinase (NEB) 1uL, T4 DNA ligase buffer 1uL, 37 DEG C of incubation 30min after mixing, after the completion plus ddH2O 9OuL to 100uL.
4) carrier digestion.The carrier structure schematic diagram is shown in Fig. 1, and sequence is shown in SEQ ID NO.5.Configure reaction system: PAc-sgRNA-Cas9 carrier 2ug, BspQI (NEB, 10U/uL) restriction endonuclease 1uL, 10xNEBuffer inscribe enzyme buffer liquid 5uL, Add ddH2O to total volume 50uL.50 DEG C are incubated for 2 hours, and then 80 DEG C of incubations, 20 minutes progress heat inactivations, glue recycle carrier-pellet Section.
5) it connects.For exon2: in 0.2mL centrifuge tube add above-mentioned steps 3) in exon2 product 2uL, add it is above-mentioned 4) step product 50ng, T4 DNA ligase 1uL and T4 DNA ligase buffer are incubated for 2 for 18 DEG C after mixing to 10uL total volume Hour.For exon13: adding above-mentioned steps 3 in 0.2mL centrifuge tube) in exon13 product 2uL, add above-mentioned 4) step product 50ng, T4 DNA ligase 1uL and T4 DNA ligase buffer are incubated for 2 hours for 18 DEG C after mixing to 10uL total volume.
6) it converts.Above-mentioned steps 5) connection product conversion DH5 α competent cell, it is coated on containing ampicillin (Ampr) in LB resistant panel, 37 DEG C of overnight incubations, picking individual colonies are added 37 DEG C of LB culture medium and cultivate 6 hours, extract weight Group plasmid.
7) it identifies.Identification recombinant plasmid is sequenced with U6Fwd primer (CGAGATCTGTTCGACTTGCAGCC).For pAc- Cas9-ABCH1.exon2-gRNA plasmid, sequencing result peak figure are shown in Fig. 2.For pAc-Cas9-ABCH1.exon2-gRNA matter Grain, sequencing result peak figure are shown in Fig. 3.Above-mentioned two carriers sequencing quality is very high, as a result reliably.
Table 1
Sequence SEQ ID No.:
exon2F TTCGGTCGCCGCCGGGCGACGAGA 5
exon2R AACTCTCGTCGCCCGGCGGCGACC 6
exon13F TTCGGACTTGCGACTACAACCCGA 7
exon13R AACTCGGGTTGTAGTCGCAAGTCC 8
PAc-Cas9-ABCH1.exon2-gRNA (SEQ ID No.:9:agatctaaaaaagcaccgactcggtgcca ctttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaactctcgtcgcccggcggc gaccgaagtattgaggaaaacatacctatataaatgatcaacatcaggaaagagcagttgagaattataagaattg gcaaatggtcttaagaaccctctgcttaagattttcaaaatttcctttagaatcaaagtgtcctattgttcgtttg ttgaaaacacagttcgatttattgactataataaattgatagttttaaatatagaggcacgactaagagagtttgg ttttgttttggttagggaccaaaaaaagtatacataacgaataaaaaaggatttaagtgcaaatggtaaaaaagtg tcgagtttttcttgagttgattgtgctgtaatgagactctgcattcggcatttgactcggcttttcctactcgtgc cgtatttcaggctgcaagtcgaacagatctcgctgcctgttatgtggcccacaaaccaagacacgttttatggcca ttaaagctggctgatcgtcgccaaacaccaaatacataatgaatatgtacacattcgagaaagaagcgatcaaaga agcgtcttcgggcggagtaggagaatgcggaggagaaggagaacgagctgatctagtatctctccacaatccaatg ccaactgaccaactggccatattcggagcaatttgaagccaatttccatcgcctggcgatcgctccattcttggct atatgtttttcaccgttacccggggccattttcaaagactcgtcggcaagataagattgtgtcactcgctgtctct cttcatttgtcgaagaatgctgaggaatttcgcgatgacgtcggcgagtattttgaagaatgagaataatttgtat ttatacgaaaatcagttagtggaattttctacaaaaacatgttatctatagataattttgttgcaaaatatgttga ctatgacaaagattgtatgtatatacctttaatgtattctcattttcttatgtatttataatggcaatgatgatac tgatgatattttaagatgatgccagaccacaggctgatttctgcgtcttttgccgaacgcagtgcatgtgcggttg ttgttttttggaatagtttcaattttcggactgtccgctttgatttcagtttcttggcttattcaaaaagcaaagt aaagccaaaaaagcgagatggcaataccaaatgcggcaaaacggtagtggaaggaaaggggtgcggggcagcggaa ggaagggtggggcggggcgtggcggggtctgtggctgggcgcgacgtcaccgacgttggagccactcctttgacca tgtgtgcgtgtgtgtattattcgtgtctcgccactcgccggttgtttttttctttttatctcgctctctctagcgc catctcgtacgcatgctcaacgcaccgcatgttgccgtgtcctttatgcgtcattttggctcgaaataggcaatta tttaaacaaagattagtcaacgaaaacgctaaaataaataagtctacaatatggttacttattgccatgtgtgtgc agccaacgatagcaacaaaagcaacaacacagtggctttccctctttcactttttgtttgcaagcgcgtgcgagca agacggcacgaccggcaaacgcaattacgctgacaaagagcagacgaagttttggccgaaaaacatcaaggcgcct gatacgaatgcatttgcaataacaattgcgatatttaatattgtttatgaagctgtttgacttcaaaacacacaaa aaaaaaaataaaacaaattatttgaaagagaattaggaatcggacagcttatcgttacgggctaacagcacaccga gacgaaatagcttacctgacgtcacagcctctggaagaactgccgccaagcagagagagagagaaaaagagggaga gcagcttagaccgcatgtgcttgtgtgtgaggcgtctctctcttcgtctcctgtttgcgcaaacgcatagactgca ctgagaaaatcgattacctattttttatgaatgaatatttgcactattactattcaaaactattaagatagcaatc acattcaatagccaaatactataccacctgagcgatgcaacgaaatgatcaatttgagcaaaaatgctgcatattt aggacggcatcattatagaaatgcttcttgctgtgtacttttctctcgtctggcagctgtttcgccgttattgtta aaaccggcttaagttaggtgtgttttctacgactagtgatgcccctactagaagatgtgtgttgcacaaatgtccc tgaataaccaatttgaagtgcagatagcagtaaacgtaagctaatatgaatattatttaactgtaatgttttaata tcgctggacattactaataaacccactataaacacatgtacatatgtatgttttggcatacaatgagtagttgggg aaaaaatgtgtaaaagcaccgtgaccatcacagcataaagataaccagctgaagtatcgaatatgagtaaccccca aattgaatcacatgccgcaactgataggacccatggaagtacactcttcatggcgatatacaagacacacacaagc acgaacacccagttgcggaggaaattctccgtaaatgaaaacccaatcggcgaacaattcatacccatatatggta aaagttttgaacgcgacttgagagcggagagcattgcggctgataaggttttagcgctaagcgggctttataaaac gggctgcgggaccagttttcatatcactaccgtttgagttcttgtgctgtgtggatactcctcccgacacaaagcc gctccatcagccagcagtcgtctaatccagagaccccggatcggggtacctactagtccagtgtggtggaattcat ggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggcccca aagaagaagcggaaggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcggca ccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacac cgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccgg ctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacg agatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacga gcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgaga aagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttcc ggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgca gacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccaga ctgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaacc tgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagct gagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctg gccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccc tgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagca gctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagcc agccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagc tgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggaga gctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatc ctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaaga gcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcg gatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcacc gtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcaga aaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaa gaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccac gatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgc tgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaa agtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgg gacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctga tccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacga gcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctc gtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagg gacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaaga acaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtac gtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaagg acgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaaga ggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaat ctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaaccc ggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgat ccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtg cgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagt accctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcga gcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattacc ctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagg gccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagac aggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggac cctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggca agtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcc catcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctg ttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgc cctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagca gaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagaga gtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagc aggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccac catcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctg tacgagacacggatcgacctgtctcagctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggcaa aaaagaaaaagggatcaggcaagcttgagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccgg ccctgctagcggtagcggcagcggtagcatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtc ccccgggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgacccggaccgcc acatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgc ggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggc ccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggc ccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgt cgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctc cccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatga cccgcaagcccggtgcctgactcgagggatccagacatgataagatacattgatgagtttggacaaaccacaacta gaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaa taaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagc aagtaaaacctctacaaatgtggtatggctgattatgatcagtcgaccgatgcccttgagagccttcaacccagtc agctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtag gacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtc gcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgag aagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctgga tggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggca ggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagccagcaaaaggccaggaaccgt aaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtc agaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgt tccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcacgc tgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgacc gctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccac tggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctac actagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgat ccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatc tcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtc atgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatat atgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttc atccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgca atgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgca gaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgcc agttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggcttca ttcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcg gtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctct tactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatg cggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgctca tcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccac tcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaat gccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagca tttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcg cacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgt atcacgaggccctttcgtcttcaaggaattaattctatattctaaaaacacaaatgatacttctaaaaaaaatcat gaatggcatcaactctgaatcaaatctttgcagatgcacctacttctcatttccactgtcacatcatttttcc)
PAc-Cas9-ABCH1.exon13-gRNA (SEQ ID No.:10:agatctaaaaaagcaccgactcggtgc cactttttcaagttgataacggactagccttattttaacttgctatttctagctctaaaactcgggttgtagtcgc aagtccgaagtattgaggaaaacatacctatataaatgatcaacatcaggaaagagcagttgagaattataagaat tggcaaatggtcttaagaaccctctgcttaagattttcaaaatttcctttagaatcaaagtgtcctattgttcgtt tgttgaaaacacagttcgatttattgactataataaattgatagttttaaatatagaggcacgactaagagagttt ggttttgttttggttagggaccaaaaaaagtatacataacgaataaaaaaggatttaagtgcaaatggtaaaaaag tgtcgagtttttcttgagttgattgtgctgtaatgagactctgcattcggcatttgactcggcttttcctactcgt gccgtatttcaggctgcaagtcgaacagatctcgctgcctgttatgtggcccacaaaccaagacacgttttatggc cattaaagctggctgatcgtcgccaaacaccaaatacataatgaatatgtacacattcgagaaagaagcgatcaaa gaagcgtcttcgggcggagtaggagaatgcggaggagaaggagaacgagctgatctagtatctctccacaatccaa tgccaactgaccaactggccatattcggagcaatttgaagccaatttccatcgcctggcgatcgctccattcttgg ctatatgtttttcaccgttacccggggccattttcaaagactcgtcggcaagataagattgtgtcactcgctgtct ctcttcatttgtcgaagaatgctgaggaatttcgcgatgacgtcggcgagtattttgaagaatgagaataatttgt atttatacgaaaatcagttagtggaattttctacaaaaacatgttatctatagataattttgttgcaaaatatgtt gactatgacaaagattgtatgtatatacctttaatgtattctcattttcttatgtatttataatggcaatgatgat actgatgatattttaagatgatgccagaccacaggctgatttctgcgtcttttgccgaacgcagtgcatgtgcggt tgttgttttttggaatagtttcaattttcggactgtccgctttgatttcagtttcttggcttattcaaaaagcaaa gtaaagccaaaaaagcgagatggcaataccaaatgcggcaaaacggtagtggaaggaaaggggtgcggggcagcgg aaggaagggtggggcggggcgtggcggggtctgtggctgggcgcgacgtcaccgacgttggagccactcctttgac catgtgtgcgtgtgtgtattattcgtgtctcgccactcgccggttgtttttttctttttatctcgctctctctagc gccatctcgtacgcatgctcaacgcaccgcatgttgccgtgtcctttatgcgtcattttggctcgaaataggcaat tatttaaacaaagattagtcaacgaaaacgctaaaataaataagtctacaatatggttacttattgccatgtgtgt gcagccaacgatagcaacaaaagcaacaacacagtggctttccctctttcactttttgtttgcaagcgcgtgcgag caagacggcacgaccggcaaacgcaattacgctgacaaagagcagacgaagttttggccgaaaaacatcaaggcgc ctgatacgaatgcatttgcaataacaattgcgatatttaatattgtttatgaagctgtttgacttcaaaacacaca aaaaaaaaaataaaacaaattatttgaaagagaattaggaatcggacagcttatcgttacgggctaacagcacacc gagacgaaatagcttacctgacgtcacagcctctggaagaactgccgccaagcagagagagagagaaaaagaggga gagcagcttagaccgcatgtgcttgtgtgtgaggcgtctctctcttcgtctcctgtttgcgcaaacgcatagactg cactgagaaaatcgattacctattttttatgaatgaatatttgcactattactattcaaaactattaagatagcaa tcacattcaatagccaaatactataccacctgagcgatgcaacgaaatgatcaatttgagcaaaaatgctgcatat ttaggacggcatcattatagaaatgcttcttgctgtgtacttttctctcgtctggcagctgtttcgccgttattgt taaaaccggcttaagttaggtgtgttttctacgactagtgatgcccctactagaagatgtgtgttgcacaaatgtc cctgaataaccaatttgaagtgcagatagcagtaaacgtaagctaatatgaatattatttaactgtaatgttttaa tatcgctggacattactaataaacccactataaacacatgtacatatgtatgttttggcatacaatgagtagttgg ggaaaaaatgtgtaaaagcaccgtgaccatcacagcataaagataaccagctgaagtatcgaatatgagtaacccc caaattgaatcacatgccgcaactgataggacccatggaagtacactcttcatggcgatatacaagacacacacaa gcacgaacacccagttgcggaggaaattctccgtaaatgaaaacccaatcggcgaacaattcatacccatatatgg taaaagttttgaacgcgacttgagagcggagagcattgcggctgataaggttttagcgctaagcgggctttataaa acgggctgcgggaccagttttcatatcactaccgtttgagttcttgtgctgtgtggatactcctcccgacacaaag ccgctccatcagccagcagtcgtctaatccagagaccccggatcggggtacctactagtccagtgtggtggaattc atggactataaggaccacgacggagactacaaggatcatgatattgattacaaagacgatgacgataagatggccc caaagaagaagcggaaggtcggtatccacggagtcccagcagccgacaagaagtacagcatcggcctggacatcgg caccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaac accgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccaccc ggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaa cgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcac gagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctga gaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagtt ccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtg cagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgcca gactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggaaa cctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcag ctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttc tggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccc cctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcag cagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggag ccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaa gctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctggga gagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaaga tcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaa gagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgag cggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttca ccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagca gaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttc aagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacatacc acgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgt gctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgac aaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatcc gggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagct gatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcac gagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagc tcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaa gggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaa gaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgt acgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaa ggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaa gaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgaca atctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaac ccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctg atccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaag tgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaa gtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagc gagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagatta ccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataa gggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcag acaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactggg accctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaaggg caagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaat cccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccc tgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccct gccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgag cagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaaga gagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagaga gcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacacc accatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcc tgtacgagacacggatcgacctgtctcagctgggaggcgacaaaaggccggcggccacgaaaaaggccggccaggc aaaaaagaaaaagggatcaggcaagcttgagggcagaggaagtcttctaacatgcggtgacgtggaggagaatccc ggccctgctagcggtagcggcagcggtagcatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacg tcccccgggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgacccggaccg ccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtc gcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcg gcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccg gcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgcc gtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacc tccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcat gacccgcaagcccggtgcctgactcgagggatccagacatgataagatacattgatgagtttggacaaaccacaac tagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgc aataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaa gcaagtaaaacctctacaaatgtggtatggctgattatgatcagtcgaccgatgcccttgagagccttcaacccag tcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgt aggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctg tcgcttgcggtattcggaatcttgcacgccctcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcg agaagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctg gatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccagg caggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaag tcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcct gttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcaatgctcac gctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccga ccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagcc actggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggct acactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaagga tctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttgg tcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtat atatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgt tcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctg caatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcg cagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcg ccagttaatagtttgcgcaacgttgttgccattgctgcaggcatcgtggtgtcacgctcgtcgtttggtatggctt cattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctcctt cggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattct cttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgta tgcggcgaccgagttgctcttgcccggcgtcaacacgggataataccgcgccacatagcagaactttaaaagtgct catcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaaccc actcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaa atgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaag catttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccg cgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggc gtatcacgaggccctttcgtcttcaaggaattaattctatattctaaaaacacaaatgatacttctaaaaaaaatc atgaatggcatcaactctgaatcaaatctttgcagatgcacctacttctcatttccactgtcacatcatttttcc)
Table 2
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>silicon Yi, a legendary monarch of Youqiong State in the xia Dynasty science and technology (Shanghai) Co., Ltd.
<120>a kind of CRISPR/Cas9 carrier that can be used for insect genes editor
<130> P2018-2396
<160> 25
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gtcgccgccg ggcgacgaga 20
<210> 2
<211> 76
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgc 76
<210> 3
<211> 96
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtcgccgccg ggcgacgaga gttttagagc tagaaatagc aagttaaaat aaggctagtc 60
cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 96
<210> 4
<211> 10663
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
ggaaaaatga tgtgacagtg gaaatgagaa gtaggtgcat ctgcaaagat ttgattcaga 60
gttgatgcca ttcatgattt tttttagaag tatcatttgt gtttttagaa tatagaatta 120
attccttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata 180
ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 240
tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 300
atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 360
attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa 420
gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 480
agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 540
aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt 600
cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat 660
cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac 720
actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg 780
cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc 840
ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa 900
ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag 960
gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct 1020
gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat 1080
ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa 1140
cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac 1200
caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc 1260
taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc 1320
cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg 1380
cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg 1440
gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca 1500
aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg 1560
cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg 1620
tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga 1680
acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac 1740
ctacagcgtg agcattgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 1800
ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc 1860
tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga 1920
tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc 1980
ctggcctttt gctggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg 2040
tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg 2100
agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag 2160
cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg 2220
gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac 2280
aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct 2340
gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata 2400
gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt 2460
cgactgatca taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc 2520
ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt 2580
attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca 2640
tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc 2700
tggatccctc gagtcaggca ccgggcttgc gggtcatgca ccaggtgcgc ggtccttcgg 2760
gcacctcgac gtcggcggtg acggtgaagc cgagccgctc gtagaagggg aggttgcggg 2820
gcgcggaggt ctccaggaag gcgggcaccc cggcgcgctc ggccgcctcc actccgggga 2880
gcacgacggc gctgcccaga cccttgccct ggtggtcggg cgagacgccg acggtggcca 2940
ggaaccacgc gggctccttg ggccggtgcg gcgccaggag gccttccatc tgttgctgcg 3000
cggccagccg ggaaccgctc aactcggcca tgcgcgggcc gatctcggcg aacaccgccc 3060
ccgcttcgac gctctccggc gtggtccaga ccgccaccgc ggcgccgtcg tccgcgaccc 3120
acaccttgcc gatgtcgagc ccgacgcgcg tgaggaagag ttcttgcagc tcggtgaccc 3180
gctcgatgtg gcggtccggg tcgacggtgt ggcgcgtggc ggggtagtcg gcgaacgcgg 3240
cggcgagggt gcgtacggcc cgggggacgt cgtcgcgggt ggcgaggcgc accgtgggct 3300
tgtactcggt catgctaccg ctgccgctac cgctagcagg gccgggattc tcctccacgt 3360
caccgcatgt tagaagactt cctctgccct caagcttgcc tgatcccttt ttcttttttg 3420
cctggccggc ctttttcgtg gccgccggcc ttttgtcgcc tcccagctga gacaggtcga 3480
tccgtgtctc gtacaggccg gtgatgctct ggtggatcag ggtggcgtcc agcacctctt 3540
tggtgctggt gtacctcttc cggtcgatgg tggtgtcaaa gtacttgaag gcggcagggg 3600
ctcccagatt ggtcagggta aacaggtgga tgatattctc ggcctgctct ctgatgggct 3660
tatcccggtg cttgttgtag gcggacagca ctttgtccag attagcgtcg gccaggatca 3720
ctctcttgga gaactcgctg atctgctcga tgatctcgtc caggtagtgc ttgtgctgtt 3780
ccacaaacag ctgtttctgc tcattatcct cgggggagcc cttcagcttc tcatagtggc 3840
tggccaggta caggaagttc acatatttgg agggcagggc cagttcgttt cccttctgca 3900
gttcgccggc agaggccagc attctcttcc ggccgttttc cagctcgaac agggagtact 3960
taggcagctt gatgatcagg tcctttttca cttctttgta gcccttggct tccagaaagt 4020
cgatgggatt cttctcgaag ctgcttcttt ccatgatggt gatccccagc agctctttca 4080
cactcttcag tttcttggac ttgccctttt ccactttggc caccaccagc acagaatagg 4140
ccacggtggg gctgtcgaag ccgccgtact tcttagggtc ccagtccttc tttctggcga 4200
tcagcttatc gctgttcctc ttgggcagga tagactcttt gctgaagccg cctgtctgca 4260
cctcggtctt tttcacgata ttcacttggg gcatgctcag cactttccgc acggtggcaa 4320
aatcccggcc cttatcccac acgatctccc cggtttcgcc gtttgtctcg atcagaggcc 4380
gcttccggat ctcgccgttg gccagggtaa tctcggtctt gaaaaagttc atgatgttgc 4440
tgtagaagaa gtacttggcg gtagccttgc cgatttcctg ctcgctcttg gcgatcatct 4500
tccgcacgtc gtacaccttg tagtcgccgt acacgaactc gctttccagc ttagggtact 4560
ttttgatcag ggcggttccc acgacggcgt tcaggtaggc gtcgtgggcg tggtggtagt 4620
tgttgatctc gcgcactttg taaaactgga aatccttccg gaaatcggac accagcttgg 4680
acttcagggt gatcactttc acttcccgga tcagcttgtc attctcgtcg tacttagtgt 4740
tcatccggga gtccaggatc tgtgccacgt gctttgtgat ctgccgggtt tccaccagct 4800
gtctcttgat gaagccggcc ttatccagtt cgctcaggcc gcctctctcg gccttggtca 4860
gattgtcgaa ctttctctgg gtaatcagct tggcgttcag cagctgccgc cagtagttct 4920
tcatcttctt cacgacctct tcggagggca cgttgtcgct cttgccccgg ttcttgtcgc 4980
ttctggtcag caccttgttg tcgatggagt cgtccttcag aaagctctga ggcacgatat 5040
ggtccacatc gtagtcggac agccggttga tgtccagttc ctggtccacg tacatatccc 5100
gcccattctg caggtagtac aggtacagct tctcgttctg cagctgggtg ttttccacgg 5160
ggtgttcttt caggatctgg ctgcccagct ctttgatgcc ctcttcgatc cgcttcattc 5220
tctcgcggct gttcttctgt cccttctggg tggtctggtt ctctctggcc atttcgatca 5280
cgatgttctc gggcttgtgc cggcccatca ctttcacgag ctcgtccacc accttcactg 5340
tctgcaggat gcccttctta atggcggggc tgccggccag attggcaatg tgctcgtgca 5400
ggctatcgcc ctggccggac acctgggctt tctggatgtc ctctttaaag gtcaggctgt 5460
cgtcgtggat cagctgcatg aagtttctgt tggcgaagcc gtcggacttc aggaaatcca 5520
ggattgtctt gccggactgc ttgtcccgga tgccgttgat cagcttccgg ctcagcctgc 5580
cccagccggt gtatctccgc cgcttcagct gcttcatcac tttgtcgtcg aacaggtggg 5640
cataggtttt cagccgttcc tcgatcatct ctctgtcctc aaacagtgtc agggtcagca 5700
cgatatcttc cagaatgtcc tcgttttcct cattgtccag gaagtccttg tccttgataa 5760
ttttcagcag atcgtggtat gtgcccaggg aggcgttgaa ccgatcttcc acgccggaga 5820
tttccacgga gtcgaagcac tcgattttct tgaagtagtc ctctttcagc tgcttcacgg 5880
tcactttccg gttggtcttg aacagcaggt ccacgatggc ctttttctgc tcgccgctca 5940
ggaaggcggg ctttctcatt ccctcggtca cgtatttcac tttggtcagc tcgttataca 6000
cggtgaagta ctcgtacagc aggctgtgct tgggcagcac cttctcgttg ggcaggttct 6060
tatcgaagtt ggtcatccgc tcgatgaagc tctgggcgga agcgcccttg tccaccactt 6120
cctcgaagtt ccagggggtg atggtttcct cgctctttct ggtcatccag gcgaatctgc 6180
tgtttcccct ggccagaggg cccacgtagt aggggatgcg gaaggtcagg atcttctcga 6240
tcttttcccg gttgtccttc aggaatgggt aaaaatcttc ctgccgccgc agaatggcgt 6300
gcagctctcc caggtggatc tggtggggga tgctgccgtt gtcgaaggtc cgctgcttcc 6360
gcagcaggtc ctctctgttc agcttcacga gcagttcctc ggtgccgtcc atcttttcca 6420
ggatgggctt gatgaacttg tagaactctt cctggctggc tccgccgtca atgtagccgg 6480
cgtagccgtt cttgctctgg tcgaagaaaa tctctttgta cttctcaggc agctgctgcc 6540
gcacgagagc tttcagcagg gtcaggtcct ggtggtgctc gtcgtatctc ttgatcatag 6600
aggcgctcag gggggccttg gtgatctcgg tgttcactct caggatgtcg ctcagcagga 6660
tggcgtcgga caggttcttg gcggccagaa acaggtcggc gtactggtcg ccgatctggg 6720
ccagcaggtt gtccaggtcg tcgtcgtagg tgtccttgct cagctgcagt ttggcatcct 6780
cggccaggtc gaagttgctc ttgaagttgg gggtcaggcc caggctcagg gcaatcaggt 6840
ttccgaacag gccattcttc ttctcgccgg gcagctgggc gatcagattt tccagccgtc 6900
tgctcttgct cagtctggca gacaggatgg ccttggcgtc cacgccgctg gcgttgatgg 6960
ggttttcctc gaacagctgg ttgtaggtct gcaccagctg gatgaacagc ttgtccacgt 7020
cgctgttgtc ggggttcagg tcgccctcga tcaggaagtg gccccggaac ttgatcatgt 7080
gggccagggc cagatagatc agccgcaggt cggccttgtc ggtgctgtcc accagtttct 7140
ttctcaggtg gtagatggtg gggtacttct cgtggtaggc cacctcgtcc acgatgttgc 7200
cgaagatggg gtgccgctcg tgcttcttat cctcttccac caggaaggac tcttccagtc 7260
tgtggaagaa gctgtcgtcc accttggcca tctcgttgct gaagatctct tgcagatagc 7320
agatccggtt cttccgtctg gtgtatcttc ttctggcggt tctcttcagc cgggtggcct 7380
cggctgtttc gccgctgtcg aacagcaggg ctccgatcag gttcttcttg atgctgtgcc 7440
ggtcggtgtt gcccagcacc ttgaatttct tgctgggcac cttgtactcg tcggtgatca 7500
cggcccagcc cacagagttg gtgccgatgt ccaggccgat gctgtacttc ttgtcggctg 7560
ctgggactcc gtggataccg accttccgct tcttctttgg ggccatctta tcgtcatcgt 7620
ctttgtaatc aatatcatga tccttgtagt ctccgtcgtg gtccttatag tccatgaatt 7680
ccaccacact ggactagtag gtaccccgat ccggggtctc tggattagac gactgctggc 7740
tgatggagcg gctttgtgtc gggaggagta tccacacagc acaagaactc aaacggtagt 7800
gatatgaaaa ctggtcccgc agcccgtttt ataaagcccg cttagcgcta aaaccttatc 7860
agccgcaatg ctctccgctc tcaagtcgcg ttcaaaactt ttaccatata tgggtatgaa 7920
ttgttcgccg attgggtttt catttacgga gaatttcctc cgcaactggg tgttcgtgct 7980
tgtgtgtgtc ttgtatatcg ccatgaagag tgtacttcca tgggtcctat cagttgcggc 8040
atgtgattca atttgggggt tactcatatt cgatacttca gctggttatc tttatgctgt 8100
gatggtcacg gtgcttttac acattttttc cccaactact cattgtatgc caaaacatac 8160
atatgtacat gtgtttatag tgggtttatt agtaatgtcc agcgatatta aaacattaca 8220
gttaaataat attcatatta gcttacgttt actgctatct gcacttcaaa ttggttattc 8280
agggacattt gtgcaacaca catcttctag taggggcatc actagtcgta gaaaacacac 8340
ctaacttaag ccggttttaa caataacggc gaaacagctg ccagacgaga gaaaagtaca 8400
cagcaagaag catttctata atgatgccgt cctaaatatg cagcattttt gctcaaattg 8460
atcatttcgt tgcatcgctc aggtggtata gtatttggct attgaatgtg attgctatct 8520
taatagtttt gaatagtaat agtgcaaata ttcattcata aaaaataggt aatcgatttt 8580
ctcagtgcag tctatgcgtt tgcgcaaaca ggagacgaag agagagacgc ctcacacaca 8640
agcacatgcg gtctaagctg ctctccctct ttttctctct ctctctgctt ggcggcagtt 8700
cttccagagg ctgtgacgtc aggtaagcta tttcgtctcg gtgtgctgtt agcccgtaac 8760
gataagctgt ccgattccta attctctttc aaataatttg ttttattttt ttttttgtgt 8820
gttttgaagt caaacagctt cataaacaat attaaatatc gcaattgtta ttgcaaatgc 8880
attcgtatca ggcgccttga tgtttttcgg ccaaaacttc gtctgctctt tgtcagcgta 8940
attgcgtttg ccggtcgtgc cgtcttgctc gcacgcgctt gcaaacaaaa agtgaaagag 9000
ggaaagccac tgtgttgttg cttttgttgc tatcgttggc tgcacacaca tggcaataag 9060
taaccatatt gtagacttat ttattttagc gttttcgttg actaatcttt gtttaaataa 9120
ttgcctattt cgagccaaaa tgacgcataa aggacacggc aacatgcggt gcgttgagca 9180
tgcgtacgag atggcgctag agagagcgag ataaaaagaa aaaaacaacc ggcgagtggc 9240
gagacacgaa taatacacac acgcacacat ggtcaaagga gtggctccaa cgtcggtgac 9300
gtcgcgccca gccacagacc ccgccacgcc ccgccccacc cttccttccg ctgccccgca 9360
cccctttcct tccactaccg ttttgccgca tttggtattg ccatctcgct tttttggctt 9420
tactttgctt tttgaataag ccaagaaact gaaatcaaag cggacagtcc gaaaattgaa 9480
actattccaa aaaacaacaa ccgcacatgc actgcgttcg gcaaaagacg cagaaatcag 9540
cctgtggtct ggcatcatct taaaatatca tcagtatcat cattgccatt ataaatacat 9600
aagaaaatga gaatacatta aaggtatata catacaatct ttgtcatagt caacatattt 9660
tgcaacaaaa ttatctatag ataacatgtt tttgtagaaa attccactaa ctgattttcg 9720
tataaataca aattattctc attcttcaaa atactcgccg acgtcatcgc gaaattcctc 9780
agcattcttc gacaaatgaa gagagacagc gagtgacaca atcttatctt gccgacgagt 9840
ctttgaaaat ggccccgggt aacggtgaaa aacatatagc caagaatgga gcgatcgcca 9900
ggcgatggaa attggcttca aattgctccg aatatggcca gttggtcagt tggcattgga 9960
ttgtggagag atactagatc agctcgttct ccttctcctc cgcattctcc tactccgccc 10020
gaagacgctt ctttgatcgc ttctttctcg aatgtgtaca tattcattat gtatttggtg 10080
tttggcgacg atcagccagc tttaatggcc ataaaacgtg tcttggtttg tgggccacat 10140
aacaggcagc gagatctgtt cgacttgcag cctgaaatac ggcacgagta ggaaaagccg 10200
agtcaaatgc cgaatgcaga gtctcattac agcacaatca actcaagaaa aactcgacac 10260
ttttttacca tttgcactta aatccttttt tattcgttat gtatactttt tttggtccct 10320
aaccaaaaca aaaccaaact ctcttagtcg tgcctctata tttaaaacta tcaatttatt 10380
atagtcaata aatcgaactg tgttttcaac aaacgaacaa taggacactt tgattctaaa 10440
ggaaattttg aaaatcttaa gcagagggtt cttaagacca tttgccaatt cttataattc 10500
tcaactgctc tttcctgatg ttgatcattt atataggtat gttttcctca atacttcgga 10560
agagcgagct cttccgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 10620
tcaacttgaa aaagtggcac cgagtcggtg cttttttaga tct 10663
<210> 5
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ttcggtcgcc gccgggcgac gaga 24
<210> 6
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
aactctcgtc gcccggcggc gacc 24
<210> 7
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ttcggacttg cgactacaac ccga 24
<210> 8
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
aactcgggtt gtagtcgcaa gtcc 24
<210> 9
<211> 10666
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
agatctaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat 60
tttaacttgc tatttctagc tctaaaactc tcgtcgcccg gcggcgaccg aagtattgag 120
gaaaacatac ctatataaat gatcaacatc aggaaagagc agttgagaat tataagaatt 180
ggcaaatggt cttaagaacc ctctgcttaa gattttcaaa atttccttta gaatcaaagt 240
gtcctattgt tcgtttgttg aaaacacagt tcgatttatt gactataata aattgatagt 300
tttaaatata gaggcacgac taagagagtt tggttttgtt ttggttaggg accaaaaaaa 360
gtatacataa cgaataaaaa aggatttaag tgcaaatggt aaaaaagtgt cgagtttttc 420
ttgagttgat tgtgctgtaa tgagactctg cattcggcat ttgactcggc ttttcctact 480
cgtgccgtat ttcaggctgc aagtcgaaca gatctcgctg cctgttatgt ggcccacaaa 540
ccaagacacg ttttatggcc attaaagctg gctgatcgtc gccaaacacc aaatacataa 600
tgaatatgta cacattcgag aaagaagcga tcaaagaagc gtcttcgggc ggagtaggag 660
aatgcggagg agaaggagaa cgagctgatc tagtatctct ccacaatcca atgccaactg 720
accaactggc catattcgga gcaatttgaa gccaatttcc atcgcctggc gatcgctcca 780
ttcttggcta tatgtttttc accgttaccc ggggccattt tcaaagactc gtcggcaaga 840
taagattgtg tcactcgctg tctctcttca tttgtcgaag aatgctgagg aatttcgcga 900
tgacgtcggc gagtattttg aagaatgaga ataatttgta tttatacgaa aatcagttag 960
tggaattttc tacaaaaaca tgttatctat agataatttt gttgcaaaat atgttgacta 1020
tgacaaagat tgtatgtata tacctttaat gtattctcat tttcttatgt atttataatg 1080
gcaatgatga tactgatgat attttaagat gatgccagac cacaggctga tttctgcgtc 1140
ttttgccgaa cgcagtgcat gtgcggttgt tgttttttgg aatagtttca attttcggac 1200
tgtccgcttt gatttcagtt tcttggctta ttcaaaaagc aaagtaaagc caaaaaagcg 1260
agatggcaat accaaatgcg gcaaaacggt agtggaagga aaggggtgcg gggcagcgga 1320
aggaagggtg gggcggggcg tggcggggtc tgtggctggg cgcgacgtca ccgacgttgg 1380
agccactcct ttgaccatgt gtgcgtgtgt gtattattcg tgtctcgcca ctcgccggtt 1440
gtttttttct ttttatctcg ctctctctag cgccatctcg tacgcatgct caacgcaccg 1500
catgttgccg tgtcctttat gcgtcatttt ggctcgaaat aggcaattat ttaaacaaag 1560
attagtcaac gaaaacgcta aaataaataa gtctacaata tggttactta ttgccatgtg 1620
tgtgcagcca acgatagcaa caaaagcaac aacacagtgg ctttccctct ttcacttttt 1680
gtttgcaagc gcgtgcgagc aagacggcac gaccggcaaa cgcaattacg ctgacaaaga 1740
gcagacgaag ttttggccga aaaacatcaa ggcgcctgat acgaatgcat ttgcaataac 1800
aattgcgata tttaatattg tttatgaagc tgtttgactt caaaacacac aaaaaaaaaa 1860
ataaaacaaa ttatttgaaa gagaattagg aatcggacag cttatcgtta cgggctaaca 1920
gcacaccgag acgaaatagc ttacctgacg tcacagcctc tggaagaact gccgccaagc 1980
agagagagag agaaaaagag ggagagcagc ttagaccgca tgtgcttgtg tgtgaggcgt 2040
ctctctcttc gtctcctgtt tgcgcaaacg catagactgc actgagaaaa tcgattacct 2100
attttttatg aatgaatatt tgcactatta ctattcaaaa ctattaagat agcaatcaca 2160
ttcaatagcc aaatactata ccacctgagc gatgcaacga aatgatcaat ttgagcaaaa 2220
atgctgcata tttaggacgg catcattata gaaatgcttc ttgctgtgta cttttctctc 2280
gtctggcagc tgtttcgccg ttattgttaa aaccggctta agttaggtgt gttttctacg 2340
actagtgatg cccctactag aagatgtgtg ttgcacaaat gtccctgaat aaccaatttg 2400
aagtgcagat agcagtaaac gtaagctaat atgaatatta tttaactgta atgttttaat 2460
atcgctggac attactaata aacccactat aaacacatgt acatatgtat gttttggcat 2520
acaatgagta gttggggaaa aaatgtgtaa aagcaccgtg accatcacag cataaagata 2580
accagctgaa gtatcgaata tgagtaaccc ccaaattgaa tcacatgccg caactgatag 2640
gacccatgga agtacactct tcatggcgat atacaagaca cacacaagca cgaacaccca 2700
gttgcggagg aaattctccg taaatgaaaa cccaatcggc gaacaattca tacccatata 2760
tggtaaaagt tttgaacgcg acttgagagc ggagagcatt gcggctgata aggttttagc 2820
gctaagcggg ctttataaaa cgggctgcgg gaccagtttt catatcacta ccgtttgagt 2880
tcttgtgctg tgtggatact cctcccgaca caaagccgct ccatcagcca gcagtcgtct 2940
aatccagaga ccccggatcg gggtacctac tagtccagtg tggtggaatt catggactat 3000
aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag 3060
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag 3120
tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag 3180
tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 3240
aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 3300
aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 3360
atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 3420
ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 3480
gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 3540
agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 3600
cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 3660
ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 3720
ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 3780
ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg 3840
agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 3900
cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 3960
cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 4020
atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 4080
tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 4140
gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 4200
ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 4260
ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 4320
ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 4380
cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 4440
accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 4500
atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 4560
ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac 4620
gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 4680
accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 4740
aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 4800
aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 4860
gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 4920
gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 4980
ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 5040
gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg 5100
aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 5160
tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt 5220
aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 5280
gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 5340
gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 5400
agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 5460
gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 5520
cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 5580
gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 5640
agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 5700
ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 5760
cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 5820
agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 5880
cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 5940
gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 6000
gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 6060
cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 6120
gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 6180
agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 6240
tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 6300
acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 6360
aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 6420
ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 6480
gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 6540
gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 6600
gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 6660
aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 6720
gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 6780
gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 6840
ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 6900
tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 6960
gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 7020
caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 7080
aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 7140
gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 7200
ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 7260
ggatcaggca agcttgaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat 7320
cccggccctg ctagcggtag cggcagcggt agcatgaccg agtacaagcc cacggtgcgc 7380
ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac 7440
taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg 7500
caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 7560
ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 7620
gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 7680
gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc 7740
gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 7800
gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 7860
ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 7920
acctggtgca tgacccgcaa gcccggtgcc tgactcgagg gatccagaca tgataagata 7980
cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 8040
aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 8100
caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag 8160
caagtaaaac ctctacaaat gtggtatggc tgattatgat cagtcgaccg atgcccttga 8220
gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac 8280
ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca 8340
ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat 8400
tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg 8460
gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac gtcttgctgg 8520
cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca 8580
tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac 8640
agcttcaagg atcgctcgcg gctcttacca gccagcaaaa ggccaggaac cgtaaaaagg 8700
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 8760
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 8820
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 8880
ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg 8940
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 9000
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 9060
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 9120
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 9180
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 9240
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 9300
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 9360
gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 9420
aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 9480
aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 9540
cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 9600
ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 9660
cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 9720
ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 9780
ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 9840
ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 9900
gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 9960
ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 10020
ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 10080
gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 10140
ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 10200
cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 10260
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 10320
aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 10380
gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 10440
gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 10500
cctataaaaa taggcgtatc acgaggccct ttcgtcttca aggaattaat tctatattct 10560
aaaaacacaa atgatacttc taaaaaaaat catgaatggc atcaactctg aatcaaatct 10620
ttgcagatgc acctacttct catttccact gtcacatcat ttttcc 10666
<210> 10
<211> 10666
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
agatctaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat 60
tttaacttgc tatttctagc tctaaaactc gggttgtagt cgcaagtccg aagtattgag 120
gaaaacatac ctatataaat gatcaacatc aggaaagagc agttgagaat tataagaatt 180
ggcaaatggt cttaagaacc ctctgcttaa gattttcaaa atttccttta gaatcaaagt 240
gtcctattgt tcgtttgttg aaaacacagt tcgatttatt gactataata aattgatagt 300
tttaaatata gaggcacgac taagagagtt tggttttgtt ttggttaggg accaaaaaaa 360
gtatacataa cgaataaaaa aggatttaag tgcaaatggt aaaaaagtgt cgagtttttc 420
ttgagttgat tgtgctgtaa tgagactctg cattcggcat ttgactcggc ttttcctact 480
cgtgccgtat ttcaggctgc aagtcgaaca gatctcgctg cctgttatgt ggcccacaaa 540
ccaagacacg ttttatggcc attaaagctg gctgatcgtc gccaaacacc aaatacataa 600
tgaatatgta cacattcgag aaagaagcga tcaaagaagc gtcttcgggc ggagtaggag 660
aatgcggagg agaaggagaa cgagctgatc tagtatctct ccacaatcca atgccaactg 720
accaactggc catattcgga gcaatttgaa gccaatttcc atcgcctggc gatcgctcca 780
ttcttggcta tatgtttttc accgttaccc ggggccattt tcaaagactc gtcggcaaga 840
taagattgtg tcactcgctg tctctcttca tttgtcgaag aatgctgagg aatttcgcga 900
tgacgtcggc gagtattttg aagaatgaga ataatttgta tttatacgaa aatcagttag 960
tggaattttc tacaaaaaca tgttatctat agataatttt gttgcaaaat atgttgacta 1020
tgacaaagat tgtatgtata tacctttaat gtattctcat tttcttatgt atttataatg 1080
gcaatgatga tactgatgat attttaagat gatgccagac cacaggctga tttctgcgtc 1140
ttttgccgaa cgcagtgcat gtgcggttgt tgttttttgg aatagtttca attttcggac 1200
tgtccgcttt gatttcagtt tcttggctta ttcaaaaagc aaagtaaagc caaaaaagcg 1260
agatggcaat accaaatgcg gcaaaacggt agtggaagga aaggggtgcg gggcagcgga 1320
aggaagggtg gggcggggcg tggcggggtc tgtggctggg cgcgacgtca ccgacgttgg 1380
agccactcct ttgaccatgt gtgcgtgtgt gtattattcg tgtctcgcca ctcgccggtt 1440
gtttttttct ttttatctcg ctctctctag cgccatctcg tacgcatgct caacgcaccg 1500
catgttgccg tgtcctttat gcgtcatttt ggctcgaaat aggcaattat ttaaacaaag 1560
attagtcaac gaaaacgcta aaataaataa gtctacaata tggttactta ttgccatgtg 1620
tgtgcagcca acgatagcaa caaaagcaac aacacagtgg ctttccctct ttcacttttt 1680
gtttgcaagc gcgtgcgagc aagacggcac gaccggcaaa cgcaattacg ctgacaaaga 1740
gcagacgaag ttttggccga aaaacatcaa ggcgcctgat acgaatgcat ttgcaataac 1800
aattgcgata tttaatattg tttatgaagc tgtttgactt caaaacacac aaaaaaaaaa 1860
ataaaacaaa ttatttgaaa gagaattagg aatcggacag cttatcgtta cgggctaaca 1920
gcacaccgag acgaaatagc ttacctgacg tcacagcctc tggaagaact gccgccaagc 1980
agagagagag agaaaaagag ggagagcagc ttagaccgca tgtgcttgtg tgtgaggcgt 2040
ctctctcttc gtctcctgtt tgcgcaaacg catagactgc actgagaaaa tcgattacct 2100
attttttatg aatgaatatt tgcactatta ctattcaaaa ctattaagat agcaatcaca 2160
ttcaatagcc aaatactata ccacctgagc gatgcaacga aatgatcaat ttgagcaaaa 2220
atgctgcata tttaggacgg catcattata gaaatgcttc ttgctgtgta cttttctctc 2280
gtctggcagc tgtttcgccg ttattgttaa aaccggctta agttaggtgt gttttctacg 2340
actagtgatg cccctactag aagatgtgtg ttgcacaaat gtccctgaat aaccaatttg 2400
aagtgcagat agcagtaaac gtaagctaat atgaatatta tttaactgta atgttttaat 2460
atcgctggac attactaata aacccactat aaacacatgt acatatgtat gttttggcat 2520
acaatgagta gttggggaaa aaatgtgtaa aagcaccgtg accatcacag cataaagata 2580
accagctgaa gtatcgaata tgagtaaccc ccaaattgaa tcacatgccg caactgatag 2640
gacccatgga agtacactct tcatggcgat atacaagaca cacacaagca cgaacaccca 2700
gttgcggagg aaattctccg taaatgaaaa cccaatcggc gaacaattca tacccatata 2760
tggtaaaagt tttgaacgcg acttgagagc ggagagcatt gcggctgata aggttttagc 2820
gctaagcggg ctttataaaa cgggctgcgg gaccagtttt catatcacta ccgtttgagt 2880
tcttgtgctg tgtggatact cctcccgaca caaagccgct ccatcagcca gcagtcgtct 2940
aatccagaga ccccggatcg gggtacctac tagtccagtg tggtggaatt catggactat 3000
aaggaccacg acggagacta caaggatcat gatattgatt acaaagacga tgacgataag 3060
atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag 3120
tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag 3180
tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 3240
aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 3300
aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 3360
atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 3420
ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 3480
gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 3540
agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 3600
cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 3660
ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 3720
ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 3780
ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg 3840
agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 3900
cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 3960
cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 4020
atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 4080
tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 4140
gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 4200
ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 4260
ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 4320
ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 4380
cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 4440
accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 4500
atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 4560
ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac 4620
gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 4680
accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 4740
aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 4800
aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 4860
gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 4920
gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 4980
ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 5040
gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg 5100
aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 5160
tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt 5220
aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 5280
gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 5340
gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 5400
agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 5460
gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 5520
cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 5580
gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 5640
agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 5700
ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 5760
cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 5820
agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 5880
cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 5940
gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 6000
gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 6060
cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 6120
gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 6180
agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 6240
tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 6300
acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 6360
aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 6420
ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 6480
gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 6540
gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 6600
gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 6660
aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 6720
gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 6780
gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 6840
ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 6900
tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 6960
gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 7020
caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 7080
aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 7140
gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 7200
ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag 7260
ggatcaggca agcttgaggg cagaggaagt cttctaacat gcggtgacgt ggaggagaat 7320
cccggccctg ctagcggtag cggcagcggt agcatgaccg agtacaagcc cacggtgcgc 7380
ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc tcgccgccgc gttcgccgac 7440
taccccgcca cgcgccacac cgtcgacccg gaccgccaca tcgagcgggt caccgagctg 7500
caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac 7560
ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc 7620
gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg 7680
gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc 7740
gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag 7800
gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc 7860
ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc 7920
acctggtgca tgacccgcaa gcccggtgcc tgactcgagg gatccagaca tgataagata 7980
cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga 8040
aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa 8100
caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag 8160
caagtaaaac ctctacaaat gtggtatggc tgattatgat cagtcgaccg atgcccttga 8220
gagccttcaa cccagtcagc tccttccggt gggcgcgggg catgactatc gtcgccgcac 8280
ttatgactgt cttctttatc atgcaactcg taggacaggt gccggcagcg ctctgggtca 8340
ttttcggcga ggaccgcttt cgctggagcg cgacgatgat cggcctgtcg cttgcggtat 8400
tcggaatctt gcacgccctc gctcaagcct tcgtcactgg tcccgccacc aaacgtttcg 8460
gcgagaagca ggccattatc gccggcatgg cggccgacgc gctgggctac gtcttgctgg 8520
cgttcgcgac gcgaggctgg atggccttcc ccattatgat tcttctcgct tccggcggca 8580
tcgggatgcc cgcgttgcag gccatgctgt ccaggcaggt agatgacgac catcagggac 8640
agcttcaagg atcgctcgcg gctcttacca gccagcaaaa ggccaggaac cgtaaaaagg 8700
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 8760
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 8820
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 8880
ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg 8940
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 9000
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 9060
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 9120
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 9180
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 9240
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 9300
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 9360
gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt 9420
aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc 9480
aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 9540
cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 9600
ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc 9660
cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta 9720
ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg 9780
ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 9840
ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 9900
gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 9960
ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga 10020
ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt 10080
gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca 10140
ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt 10200
cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 10260
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 10320
aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt 10380
gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc 10440
gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa 10500
cctataaaaa taggcgtatc acgaggccct ttcgtcttca aggaattaat tctatattct 10560
aaaaacacaa atgatacttc taaaaaaaat catgaatggc atcaactctg aatcaaatct 10620
ttgcagatgc acctacttct catttccact gtcacatcat ttttcc 10666
<210> 11
<211> 15
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
atgcttataa cggag 15
<210> 12
<211> 87
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
agctcgccgc cgtcgccgcc gggcgacgag aaggagtcca agatgagcca gatacagttc 60
tcacagccct tcgacgagga cgcctcg 87
<210> 13
<211> 119
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
gtgtggtcga agcggcaaca ggcggtgtgc gtgagacacg cctacaagca ctacggctcc 60
aacaagcggc ccaaccacgt gctcagcaac ctcaacatga ccgtcgccaa ggggaccat 119
<210> 14
<211> 119
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ctacggtctg ctgggcgcgt cgggctgcgg caagacgacg ctgctgtcgt gcatcgtggg 60
gcgccgcaag ctcaacagcg gggagatctg ggtgctgggc ggcaagcccg ggactaaag 119
<210> 15
<211> 196
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gctcgggcgt gccgggcaag cgcgtgggct acatgccgca ggagatcgcg ctgtacggag 60
agttcaccat caaggagacc atgatgtact tcggctggat cttcggcatg aagacgtcgg 120
agatcctgga gcggctgcgc ttcctgctgg acttcctcga cctgcccagc gagaacagga 180
tggtcaagaa cctcag 196
<210> 16
<211> 202
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
cggcggtcag tcgcggcgcg tgtcgttcgc ggtggcgctg atgcacgacc ccgagctgct 60
gatcctggac gagcccacgg tgggcgtgga cccgctgctg cgccagtcca tctggacgca 120
cctcgtgcgc atcaccggcg ccgccgacaa gaccgtcatc atcaccacgc actacatcga 180
ggaggcgcgc caggcgcaca cc 202
<210> 17
<211> 115
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ataggtctga tgcggagcgg gcggctgctg gcggagcagt ctcctcaggc gctgctgcag 60
atgtacaact gcatctcact cgaagacgtc ttcctcaagc tgtcccggaa acagg 115
<210> 18
<211> 176
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
caggtcagtc caaccaggcg gtagagctcc aagtggcggg cgggctcggg ctcaccaagc 60
tgtcgcggcg cgaggaggcg ccctacgccc acgaggatgc gcacgtcaag gggctcaact 120
tccaccagag caaggaggtg ctcatcacgg accacaatgg cgttagtaat ggcgat 176
<210> 19
<211> 63
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
attcccggca agatcgcgcc gagcatgaag gccgactgtg atgactgcgg agactgcttg 60
agt 63
<210> 20
<211> 168
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ccactcctga atttcacctc ccgagggaag atcaaggcgc tgatccagaa gaacttcctg 60
cgcatgtgga ggaacatcgg ggtgatgctg ttcatcttcg ttctgcccgt catgcaggtc 120
atcctgttct gtctcgccat cggacgggac cccgtcgggc tgagactg 168
<210> 21
<211> 235
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
gcgatagtaa acaacgacgt gaacgtagtg gacggctact gtccgtacaa caacacgtgc 60
tccatgaaga acctgtcctg ccgctacctc gaccacctca agaaccagtc catcatcaag 120
gagtactacg ccgacctcga aagcgccatc aacgccgtca aggaggggca ggcgtggggc 180
gccatctact tcaatgagaa ttatacagac tctctggtcg cgagactggc tttgg 235
<210> 22
<211> 125
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
ctgacactgc ggacgacgag accatcgagt cgtcagaagt gcaggtgtgg ctcgacatgt 60
ccaaccagca gatcggcctc atgctcaacc gagacataca gttctcatac agggacttcg 120
ctaag 125
<210> 23
<211> 125
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
aacttgctgt cgacttgcga ctacaacccg aaggtgggcg acatcccgat cgacttcaag 60
gagcccatct acggcgacac caacccctcc ttcactgact tcgtcgctcc cggtgttatt 120
ctcac 125
<210> 24
<211> 245
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tatcgtgttc ttcctggcgg tggcgctgac gtcgtcggcg ctgatcgtgg agcgcatgga 60
ggggctgctg gaccgctcgt gggtggccgg cgtgtccccc ggggagatcc tgttctcgca 120
cgtcgtcacg cagttcgtcg tcatgtgcgg ccagaccgcg ctcgtgctcg tgttcatgat 180
actggtgttc ggcgtcaagt gtaacgggaa tatcttcttt gtggtcctgt tgacgctgct 240
gcaag 245
<210> 25
<211> 323
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gtctgtgcgg gatgtgcttc ggcttcgtaa tctccgccgc gtgcgaactg gaacgaaacg 60
cgatccagct agcactcggc tcattctacc ccacgcttct tctgagcggg gtgatctggc 120
ccatcgaggg catgccgtgg atcctgcgct acatctccac gtgtctgccg ctgactctgg 180
cgacctcctc gctgcggtcc atcctgaccc gcggctggcc catcaccgac cccgaggtct 240
acatgggctt catctccacc ctcatctgga tcgctctgtt cctcgtagtc accatcacag 300
ttctaaggtt caagaaagga taa 323

Claims (10)

1. a kind of for editing the reagent of insect cell gene, the reagent includes:
(i) sgRNA of ABC transporter gene or the expression vector for expressing the sgRNA are targeted；With
The expression cassette of optional (ii) expression Cas9 albumen or expression Cas9 albumen.
2. reagent as described in claim 1, which is characterized in that the ABC transporter gene is ABCH1.
3. reagent as described in claim 1, which is characterized in that the sgRNA identification region sequence has SEQ ID NO.:1 institute The nucleotide sequence shown.
4. a kind of kit, the kit contains:
(1) the first container and the first expression vector in the first container, first expression vector contain first Expression cassette, first expression cassette are used to express the sgRNA of the targeting ABC transporter gene；With
Optional (2) second container and the second expression vector in the second container, second expression vector contain There is the second expression cassette, second expression cassette is used to express the expression cassette of Cas9 albumen.
5. a kind of method for carrying out gene editing in insect cell, which comprises
Insect cell is handled with reagent described in claim 1, to carry out gene editing.
6. the purposes of reagent as described in claim 1, the reagent is used to prepare kit, and the kit is used for: (a) being struck Except ABC transporter gene；And/or (b) for editing insect cell gene.
7. a kind of sgRNA, the sgRNA of the sgRNA targeting ABC transporter gene.
8. a kind of carrier, the carrier includes sgRNA as claimed in claim 7.
9. a kind of gene editing system, the gene editing system includes sgRNA as claimed in claim 7.
10. a kind of disinsection method, which comprises
Insect cell is handled with reagent described in claim 1, to carry out gene editing.
CN201910156588.2A 2019-03-01 2019-03-01 A kind of CRISPR/Cas9 carrier can be used for insect genes editor Pending CN109666702A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110760514A (en) * 2019-10-31 2020-02-07 福建农林大学 Species-specific insecticides based on genome editing and DNA barcodes

Citations (1)

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CN104610440A (en) * 2015-02-15 2015-05-13 中国农业科学院蔬菜花卉研究所 ABC transportprotein gene ABCH1 and application of specific dsRNA of ABCH1 in prevention and control of diamond back moth and Bt resistance treatment

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CN104610440A (en) * 2015-02-15 2015-05-13 中国农业科学院蔬菜花卉研究所 ABC transportprotein gene ABCH1 and application of specific dsRNA of ABCH1 in prevention and control of diamond back moth and Bt resistance treatment

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DENECKE S.等: "Describing the role of Drosophila melanogaster ABC transporters in insecticide biology using CRISPR-Cas9 knockouts", 《INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY》 *
张婷婷等: "RNA靶向基因编辑技术CRISPR/Cas9在昆虫中的研究进展", 《天津师范大学学报（自然科学版）》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110760514A (en) * 2019-10-31 2020-02-07 福建农林大学 Species-specific insecticides based on genome editing and DNA barcodes
CN110760514B (en) * 2019-10-31 2021-06-01 福建农林大学 Species-specific insecticides based on genome editing and DNA barcodes

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