CN107164402A - A kind of gene editing carrier and its application based on CRISPR Cas9 systems - Google Patents

Abstract

The invention provides a kind of genome editor carrier based on CRISPR Cas9 systems and its application, belong to plant genetic engineering field, and in particular to a kind of Wheat volatiles editor carrier and its application based on CRISPR Cas9 systems.Conversion carrier of the invention by comparing CRISPR Cas9 systems, using gene editing efficiency when different Cas9 promoters, difference sgRNA promoters and difference sgRNA sequential structures, obtains the higher CRISPR Cas9 conversion carriers of the editorial efficiency in wheat.This optimization carrier significantly improves the efficiency that wheat crop carries out gene editing using CRISPR Cas9, overcome the problem of mutation rate is low after the conversion of CRISPR Cas9 carriers, acquisition CRISPR Cas9 transgenosis mutating strain series are difficult in the crops such as wheat, transformation time is saved, conversion cost is reduced, more preferable basis has been established to popularization and application of the CRISPR Cas9 gene editing systems in crop.

Description

It is a kind of based on the gene editing carrier of CRISPR-Cas9 systems and its application

Technical field

The invention belongs to plant genetic engineering field, and in particular to a kind of wheat cdna based on CRISPR-Cas9 systems Group editor's carrier and its application.

Background technology

From the end of the eighties in last century, the genetic modification technology based on artificial endonucleases starts development, at present main bag Include：First generation artificial endonucleases Zinc finger nuclease (ZFN) technology, second generation artificial endonucleases' transcriptional activation increment effect Answer thing nuclease (TALEN) technology, third generation artificial endonucleases' CRISPR-Cas9 nucleic acid zymotechnics.ZFN technologies are specifically known , easily there is phenomenon of missing the target in other ability, and causes other purposes gene mutation and chromosome aberration.In addition, ZFN design screening Time and effort consuming, cost is high, therefore limits it and be more widely applied.TALEN is small with respect to the ZFN technologies probability that misses the target, cell toxicant Property is small, is once widely used.As emerging gene editing technology, CRISPR-Cas9 has unrivaled advantage: 1. it is higher to target accuracy.RNA target must be matched completely to sequence and genome sequence, and Cas9 can just be sheared to DNA.2. It can be achieved to knock out the multiple sites of target gene simultaneously.3. experimental period is short, efficiency high.4. target spot is more, no species limitation.

The allohexaploid that wheat is made up of tri- sets of genomes of A, B, D, the average copy number of gene is 2.8, wherein Gene (46%) close to half has a 3-4 copy, and 12% gene has a 1-2 copy, 42% gene copy number >=5, Therefore in the urgent need to setting up the gene stacking transgeneic procedure system of recombinase-mediated in wheat, DNA superpositions/deletion is realized, And the system is utilized, target strain of the exploitation with independent intellectual property right in wheat.With traditional genome editing technique ZFN Compared with TALEN, using CRISPR-Cas9 rite-directed mutagenesises, cost is lower, it is easier to operate, more efficient, it is easier to obtain pure Zygotic mutation body, the research to wheat cdna function is significant.

Inst. of Genetics and Development Biology, CAS is engaged in high rosy clouds laboratory Plant Genome fixed point editor's skill Art research, establishes the nuclease-mediated site-directed point mutation technologies of TALEN in the plant species such as two fringe false bromegrass, paddy rice System.Realize that TaMLO-A plants in bread wheat protoplast and transgenosis using CRISPR-Cas9 systems in the laboratory in 2014 Rite-directed mutagenesis in strain.The carrier of CRISPR-Cas9 gene editing systems contains two expression cassettes --- sgRNA expression cassettes and Cas9 expression cassettes, a kind of nuclease of Cas9 gene codes of Cas9 expression cassettes expression, what sgRNA expression cassettes were expressed carries target sequence SgRNA, guiding Cas9 nucleases the position of target sequence is sheared by DNA double chain on genome.Cas9 genes and sgRNA Expression and sgRNA sequential structure, can all influence gene editing efficiency.

The present invention is based on above technical background, by comparing the conversion carrier of CRISPR-Cas9 systems, using different Cas9 Gene editing efficiency when promoter, difference sgRNA promoters and difference sgRNA sequential structures, obtains and is edited in wheat More efficient CRISPR-Cas9 conversion carriers.This optimization carrier is significantly improved wheat crop and carried out using CRISPR-Cas9 The efficiency of gene editing, overcomes in the crops such as wheat after the conversion of CRISPR-Cas9 carriers that mutation rate is low, obtain CRISPR- The problem of Cas9 transgenosis mutating strain series are difficult, has saved transformation time, has reduced conversion cost, and CRISPR-Cas9 genes are compiled Collect popularization and application of the system in crop and establish more preferable basis.

The content of the invention

All bibliography being mentioned herein all are incorporated herein by reference.

Unless there are conversely indicating, all technologies used herein and scientific terminology are all with common with art of the present invention The identical implication that technical staff is generally understood.Unless there are conversely indicating, technology that is used herein or mentioning is ability Standard technique known to the those of ordinary skill of domain.Material, method and example are only used as to illustrate, rather than are any limitation as.

, can be extensive the invention provides a kind of more efficient CRISPR-Cas9 gene editing systems of gene editing Applied to different crops, the crop includes but is not limited to corn, wheat, rape, paddy rice, sorghum, soybean, barley and millet Deng.

In order to improve the gene editing efficiency in crop, the present invention is to the sgRNA sequential structures in CRISPR-Cas9 systems Transformed and optimized, improved sgRNA structures are named as sgRNA4TmC+5, its nucleotide sequence such as SEQ ID NO: Shown in 20.Experiment finds that sgRNA structures are sgRNA4TmC+5 (SEQ ID NO:20) the CRRISPR/Cas9 systems of structure exist It is transferred to after wheat plant, its gene editing efficiency is better than traditional sgRNA structures and tRNA-gRNA structures.

In CRISPR-Cas9 gene editings carrier provided by the present invention, one is also connected with before sgRNA4TmC+5 structures Promoter, the promoter includes but is not limited to the promoters such as TaU3p, OsU3p, TaU6 and OsU6bp, is preferably that TaU3 starts Son.Above-mentioned CRISPR-Cas9 gene editings system is also comprising a Cas9 gene, and the nucleotide sequence of the Cas9 genes is such as SEQ ID NO:Shown in 5.More specifically, operability is connected with a promoter and drives its table before the Cas9 genes Reach, the promoter includes but is not limited to Ubi and 2*35S promoters, preferably Ubi promoters.

Present invention also offers a kind of CRISPR-Cas9 edit methods of plant gene, sgRNA structures are in methods described SgRNA4TmC+5, its nucleotide sequence such as SEQ ID NO:Shown in 20.

One is also connected with CRISPR-Cas9 gene editings method provided by the present invention, before sgRNA4TmC+5 structures to open Mover, the promoter includes but is not limited to the promoters such as TaU3p, OsU3p, TaU6 and OsU6bp, is preferably TaU3 promoters.

Specifically, CRISPR-Cas9 gene editings method is also comprising a Cas9 gene, the nucleotides of the Cas9 genes Sequence such as SEQ ID NO:Shown in 5.More specifically, operability is connected with a promoter driving before the Cas9 genes It is expressed, and the promoter includes but is not limited to Ubi and 2*35S promoters, preferably Ubi promoters.

CRISPR-Cas9 gene editings carrier provided by the present invention and method, can apply to the base to different plants Because a group DNA enters edlin, the editor include but be not limited by the system so that plant genome DNA occur base deletion or Insertion；Also include can apply to the fields such as conditional gene knockout, gene knock-in, gene replacement, point mutation.

Present invention also offers a kind of method for obtaining wheat anti-powdery mildew mutant, methods described uses CRISPR- Cas9 gene editing methods, it is characterised in that the target sequence of the gene editing system such as SEQ ID NO:Shown in 2.

The method provided by the present invention for obtaining wheat anti-powdery mildew mutant, wherein described CRISPR-Cas9 genes SgRNA structures in edit methods are sgRNA4TmC+5 structures, the nucleotide sequence such as SEQ of the sgRNA4TmC+5 structures ID NO:Shown in 20.

Compared to prior art, the advantage of CRISPR-Cas9 gene editings method provided by the present invention and carrier is：It is aobvious The editorial efficiency for improving wheat CRISPR-Cas9 is write, overcomes after wheat CRISPR-Cas9 carriers are converted that mutation rate is low, obtain The problem of CRISPR-Cas9 transgenosis mutating strain series are difficult, has saved transformation time, has reduced conversion cost.

Brief description of the drawings

Fig. 1 is p286 and p294 carrier structure schematic diagrames.TaMLO-A is the target sequence on wheat TaMLO-A genes, SgRNA is single guide RNA coded sequences, and Amp is carrier amicillin resistance site.

Fig. 2 is p286 and p294 vector gene editorial efficiency comparative result figures.Plasmid illustrates for plasmid, and Dra II are Restriction enzyme, " +/- " is respectively add/not add the restriction endonucleases of Dra II, and Mutation rate are mutation efficiency.

Fig. 3 is p342, p338, p295 and p341 carrier structure schematic diagram.Target sequence on L475 Wheat volatiles, SgRNA is single guide RNA coded sequences, and Amp is carrier amicillin resistance site.

Fig. 4 is p342, p338, p295 and p341 vector gene editorial efficiency comparative result figure.Plasmid shows for plasmid Meaning, BanI is restriction enzyme, and " +/- " is respectively add/not add BanI restriction endonucleases, and Mutation rate are imitated for mutation Rate.

Fig. 5 is p345 and p349 carrier structure schematic diagrames.Target sequence on L475 Wheat volatiles, sgRNA is single Guide RNA coded sequences, sgRNA4TmC+5 is the single guide RNA coded sequences of optimization, and tRNA is tRNA^GlyCompile Code sequence, Amp is carrier amicillin resistance site.

Fig. 6 is p342, p345 and p349 vector gene editorial efficiency comparative result figures.Plasmid illustrates for plasmid, BanI For restriction enzyme, " +/- " is respectively add/not add BanI restriction endonucleases, and Mutation rate are mutation efficiency.

Embodiment

With reference to specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following implementation Example.In the present invention unless otherwise noted, raw material is commercially available obtains, and method is the conventional method of this area.

Embodiment one：Select the promoter for driving Cas9 genes

This experiment using one section of nucleotide sequence on gene of wheat powdery mildew TaMLO-A genes as target sequence, by than The gene editing efficiency of CRISPR-Cas9 systemic vectors when driving Cas9 gene expressions compared with different promoters, optimizes CRISPR- The carrier of Cas9 systems.

1. carrier explanation

P286 carriers and p294 carriers are comprising two expression cassettes --- sgRNA expression cassettes and Cas9 expression cassettes, its difference It is that the promoter of Cas9 expression cassettes is different, p286 carriers drive the expression of Cas9 genes, p294 carriers using 2*35s promoters The expression of Cas9 genes is driven using ubi promoters.Specifically：

P286 carriers include TaU6p::TaMLO-A-sgRNA expression cassettes and 2*35s::Cas9 expression cassettes.TaU6p:: TaMLO-A-sgRNA expression cassettes element includes TaU6p promoters (SEQ ID NO:1), the target sequence (SEQ on TaMLO-A genes ID NO:2) with sgRNA scaffold (SEQ ID NO:3)；TaU6p promoters derive from wheat CB037, TaMLO-A gene On target sequence and sgRNA scaffold be according to article announce the artificial synthesized fragment of sequence.2*35s::Cas9 expression cassette bags Include 2*35s promoters (SEQ ID NO:4), Cas9 fragments (SEQ ID NO:5) with CaMV terminators (SEQ ID NO:6)；2* It is to announce sequence according to article manually to close that 35s promoters and CaMV terminators, which are respectively derived from plasmid pGIGI and pLGZ2, Cas9, Into fragment (Yanpeng Wang, Xi Cheng, Qiwei Shan, Yi Zhang, Jinxing Liu, Caixia Gao＆Jin- Long Qiu.Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.Nat.biotech.2014,32:947-951)。 P286 carrier structures are shown in Figure 1A.

P294 carriers include TaU6p::TaMLO-A-sgRNA and Ubi::Two expression cassettes of Cas9.TaU6p::TaMLO-A- SgRNA expression cassettes are as it was previously stated, Ubi::Cas9 expression cassettes include Ubi promoters (SEQ ID NO:7), Cas9 genes (SEQ ID NO:5) with CaMV terminators (SEQ ID NO:6), Ubi promoters derive from wheat cdna rifle routine transformation carrier PAHC20. P294 carrier structures are shown in Figure 1B.

2. the gene editing efficiency comparison of different carriers

Using PEG methods by p286, p294, GFP plasmid transformed wheat Leaves Protoplast, GFP plasmids are used as protoplast The positive control of conversion and the negative control of gene editing.After 24 DEG C are cultivated 48 hours, protoplast is collected.

Using PCR/RE (Polymerase Chain Reaction/Restriction digestion) method to difference The gene editing efficiency of plasmid is detected and compared.

First, the genomic DNA of protoplast is extracted, TaMLO-A fragments is expanded, is detected through agarose gel electrophoresis, is expanded Increase purpose band single.Amplimer is as follows：

P1：5’-TGGCGCTGGTCTTCGCCGTCATGATCATCGTC-3’(SEQ ID NO:8)

P2：5’-TACGATGAGCGCCACCTTGCCCGGGAA-3’(SEQ ID NO:9)

Then, PCR primer is carried out using restriction enzyme DraII (restriction enzyme site included in target sequence) Digestion, and electrophoresis detection.As a result it is as shown in Figure 2：As the GFP samples of gene editing negative control, its PCR primer is by complete enzyme Cut, and the PCR primer for converting p286 and p294 plasmids can not be cut completely through.Calculated by ImageJ softwares and do not cut DNA Fragment accounts for the mutation efficiency of the percentage of digestion STb gene amount, the i.e. sample.It is computed, the mutation rate of p286 samples is 2.6%, The mutation rate of p294 samples is that 7.6%, p294 samples mutation efficiency is higher than p286 samples, therefore is turned in wheat CRISPR-Cas9 In change system, Ubi-Cas9 editorial efficiencies are better than 2*35SCas9.Illustrate in the CRISPR-Cas9 gene editings system for wheat In system, with the driving Cas9 expression of Ubi promoters, the editorial efficiency of CRISPR-Cas9 gene editing systems can be improved.

Embodiment two selects the promoter for driving sgRNA to express

In order to further improve wheat CRISPR-Cas9 editorial efficiency, we compare in wheat protoplast system During different snoRNA promoters driving sgRNA expression, editorial efficiency of the CRISPR-Cas9 carriers to Wheat volatiles sequence. At presentPlantIn be usually used in drive sgRNA expression snoRNA promoters have TaU3p (SEQ ID NO:10)、OsU3p(SEQ ID NO:11)、TaU6(SEQ ID NO:1) with OsU6bp (SEQ ID NO:12), it is also several startups that this experiment emphasis compares Son.

1. carrier explanation

P342 carriers (Fig. 3 A), p338 carriers (Fig. 3 B), p295 carriers (Fig. 3 C) and p341 carriers (Fig. 3 D) include two Individual expression cassette --- sgRNA expression cassettes and Cas9 expression cassettes.Cas9 expression cassettes include Ubi promoters (SEQ ID NO:7)、 Cas9 fragments (SEQ ID NO:5) with CaMV terminators (SEQ ID NO:6), target sequence is Wheat volatiles sequence L475 (SEQ ID NO:13), its difference is to drive the promoter of sgRNA expression different.P343 carriers are TaU3 promoters (SEQ ID NO:10), from Chinese spring；P339 carriers are OsU3 promoters (SEQ ID NO:11), derive from 11 are spent in paddy rice；P340 carriers are TaU6 promoters (SEQ ID NO:1), from wheat CB037；P333 carriers It is OsU6b promoters (SEQ ID NO:12), from Nipponbare paddy rice.

2. the gene editing efficiency comparison of different carriers

Using PEG methods by p342, p338, p295, p341 and GFP plasmid transformed wheat Leaves Protoplast, GFP plasmids are made The negative control of positive control and gene editing for protoplast transformation.After 24 DEG C are cultivated 48 hours, protoplast is collected.Profit With gene of PCR/RE (the Polymerase Chain Reaction/Restriction digestion) methods to different plasmids Editorial efficiency is detected and compared, and is comprised the following steps that：

First, the genomic DNA of protoplast is extracted, amplification includes target sequence L475 DNA fragmentation.Due to wheat be by The allohexaploid of tri- sets of genome compositions of A, B, D, three sets of genomes include target sequence L475 (SEQ ID NO.13), therefore Need to expand DNA fragmentation --- L475A, L475B and the L475D for including target sequence L475 in three sets of genomes respectively.Amplification is drawn Thing is as follows：

P3：5’-TTCGGGGTTTTGCATGTCAGCTAGTACGGAG-3’(SEQ ID NO:14)

P4：5’-GTGGACACGAACCGCTGC-3’(SEQ ID NO:15)

P5：5’-GACGCTGTGATGATCAATGGTGCCGTG-3’(SEQ ID NO:16)

P6：5’-AGCGCGTCCGTGAAGTGCTCCTGGTTC-3’(SEQ ID NO:17)

P7：5’-GACGCTGTGATGACCAATGGTGCCATAC-3’(SEQ ID NO:18)

P8：5’-CGACGTGTCGGCCAGCGCA-3’(SEQ ID NO:19)

Wherein P3 and P4 is used for L475A amplification, and P5 and P6 are used for L475B amplification, and P7 and P8 are used for L475D expansion Increase.Detected through agarose gel electrophoresis, amplification purpose band is single.

Then, PCR primer is carried out using restriction enzyme BanI (restriction enzyme site included in target sequence) Digestion, and electrophoresis detection.As a result it is as shown in Figure 4：As the GFP samples of gene editing negative control, its PCR primer is by complete enzyme Cut, and the PCR primer for converting p342, p338, p295 and p341 plasmid can not be cut completely through.Calculated by ImageJ softwares The mutation efficiency of the percentage that DNA fragmentation accounts for digestion STb gene amount, the i.e. sample is not cut.Be computed, L475A mutation rate according to Secondary is 10.4%, 4.5%, 0.2%, 4.6%；L475B mutation rate is followed successively by 11.3%, 4.2%, 0,3.0%；L475D's Mutation rate is followed successively by 9.1%, 3.0%, 0.3%, 3.6%.Illustrate in the CRISPR-Cas9 gene editing systems for wheat In, sgRNA expression is driven with TaU3 promoters, the editor's effect that can further improve CRISPR-Cas9 gene editing systems Rate.

The screening of the sgRNA of embodiment three difference sequential structures

In paddy rice CRRISPR-Cas9 gene editing systematic researches, sgRNA is expressed using tRNA re-recording systems, can be with Expressing for sgRNA is improved, so as to improve CRRISPR-Cas9 gene editing efficiency；And sent out in the research of human cell Existing, CRISPR-Cas9 systems use gene editing efficiency difference during different sgRNA sequential structures very big.In wheat, also The research not influenceed on sgRNA expression ways and its sequential structure on gene editing efficiency and report.In order to further excellent Change the carrier of wheat CRISPR-Cas9 systems, improve the editorial efficiency to Wheat volatiles sequence, we are with Wheat volatiles sequence Row L475 is used as target sequence (SEQ ID NO:13), to the gene of the CRISPR-Cas9 carriers containing different sgRNA sequential structures Editorial efficiency is compared.

1. carrier explanation

P342 carriers (Fig. 3 A), p345 carriers (Fig. 5 A) and p349 carriers (Fig. 5 B) and p341 carriers (Fig. 3 D) include two Individual expression cassette --- sgRNA expression cassettes and Cas9 expression cassettes.Cas9 expression cassettes include Ubi promoters (SEQ ID NO:7)、 Cas9 fragments (SEQ ID NO:5) with CaMV terminators (SEQ ID NO:6), target sequence is Wheat volatiles sequence L475 (SEQ ID NO.13), the promoter of driving sgRNA expression is TaU3 promoters, and it distinguishes the sequential structure for being sgRNA It is different.SgRNA in p342 carriers is sgRNA sequences (SEQ ID NO the most frequently used at present：3)；SgRNA in p345 carriers For sgRNA4TmC+5 (SEQ ID NO:20, artificial synthesized)；TRNA (SEQ ID NO are added before sgRNA in p349 carriers: 21, from Chinese spring), target sequence is located between tRNA and sgRNA.

2. the gene editing efficiency comparison of different carriers

Using PEG methods by p342, p345, p349 and GFP plasmid transformed wheat Leaves Protoplast, GFP plasmids are used as original The positive control of raw plastid transformation and the negative control of gene editing.After 24 DEG C are cultivated 48 hours, protoplast is collected.Utilize PCR/RE (Polymerase Chain Reaction/Restriction digestion) methods are compiled to the gene of different plasmids Collect efficiency to be detected and compared, comprise the following steps that：

First, the genomic DNA of protoplast is extracted, amplification includes target sequence L475 DNA fragmentation.Due to wheat be by The allohexaploid of tri- sets of genome compositions of A, B, D, three sets of genomes include target sequence L475, it is therefore desirable to expand respectively Target sequence L475 DNA fragmentation --- L475A, L475B and L475D is included in three sets of genomes.Amplimer be the same as Example two, Detected through agarose gel electrophoresis, amplification purpose band is single.

Then, PCR primer is carried out using restriction enzyme BanI (restriction enzyme site included in target sequence) Digestion, and electrophoresis detection.As a result it is as shown in Figure 6：As the GFP samples of gene editing negative control, its PCR primer is by complete enzyme Cut, and the PCR primer for converting p342, p345 and p349 plasmid can not be cut completely through.Calculated and do not cut by ImageJ softwares Open the mutation efficiency of the percentage that DNA fragmentation accounts for digestion STb gene amount, the i.e. sample.It is computed, L475A mutation rate is followed successively by 12.1%th, 31.8%, 8.2%；L475B mutation rate is followed successively by 11.6%, 22.8%, 7.8%；L475D mutation rate is successively For 7.7%, 19.7%, 4.9%.Illustrate in the CRISPR-Cas9 gene editing systems for wheat, sgRNA is (SEQ ID NO during sgRNA4TmC+5 sequences：20) editorial efficiency of CRISPR-Cas9 systems can, be significantly improved.

In summary, the promoter of driving Cas9 gene expressions is compared present system, drive opening for sgRNA expression Influence of mover and the sgRNA sequential structure to wheat cdna editorial efficiency, as a result shows to be better than 2*35S using Ubi promoters Promoter, TaU3 promoters are better than OsU3 promoters, TaU6 promoters and OsU6b promoters, and sgRNA4TmC+5 is better than traditional SgRNA and tRNA-gRNA structures.Summary result, the final optimization carrier TaU3p for obtaining wheat CRRISPR-Cas9:: sgRNA4TmC+5-Ubi::Cas9-PUC19.This optimization carrier will significantly improve wheat CRISPR-Cas9 editorial efficiency, overcome Wheat CRISPR-Cas9 conversions mutation rate is low, the problem of CRISPR-Cas9 transgenosis mutating strain series are difficult is obtained, when saving conversion Between, reduce conversion cost.

SEQUENCE LISTING

<110>Non- name prosperous system Crop Design forward position laboratory（Beijing）Co., Ltd

<120>It is a kind of based on the gene editing carrier of CRISPR-Cas9 systems and its application

<130>

<160> 21

<170> PatentIn version 3.3

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acgttgtttg aggataggga aatgatcgag gaacgcttga agacgtatgc ccatctcttc 1980

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atcgaggaag gcattaagga gctggggagt cagatcctta aggagcaccc ggtggaaaac 2460

acgcagttgc aaaatgagaa gctctatctg tactatctgc aaaatggcag ggatatgtat 2520

gtggaccagg agttggatat taaccgcctc tcggattacg acgtcgatca tatcgttcct 2580

cagtccttcc ttaaggatga cagcattgac aataaggttc tcaccaggtc cgacaagaac 2640

cgcgggaagt ccgataatgt gcccagcgag gaagtcgtta agaagatgaa gaactactgg 2700

aggcaacttt tgaatgccaa gttgatcaca cagaggaagt ttgataacct cactaaggcc 2760

gagcgcggag gtctcagcga actggacaag gcgggcttca ttaagcggca actggttgag 2820

actagacaga tcacgaagca cgtggcgcag attctcgatt cacgcatgaa cacgaagtac 2880

gatgagaatg acaagctgat ccgggaagtg aaggtcatca ccttgaagtc aaagctcgtt 2940

tctgacttca ggaaggattt ccaattttat aaggtgcgcg agatcaacaa ttatcaccat 3000

gctcatgacg catacctcaa cgctgtggtc ggaacagcat tgattaagaa gtacccgaag 3060

ctcgagtccg aattcgtgta cggtgactat aaggtttacg atgtgcgcaa gatgatcgcc 3120

aagtcagagc aggaaattgg caaggccact gcgaagtatt tcttttactc taacattatg 3180

aatttcttta agactgagat cacgctggct aatggcgaaa tccggaagag accacttatt 3240

gagaccaacg gcgagacagg ggaaatcgtg tgggacaagg ggagggattt cgccacagtc 3300

cgcaaggttc tctctatgcc tcaagtgaat attgtcaaga agactgaagt ccagacgggc 3360

gggttctcaa aggaatctat tctgcccaag cggaactcgg ataagcttat cgccagaaag 3420

aaggactggg acccgaagaa gtatggaggt ttcgactcac caacggtggc ttactctgtc 3480

ctggttgtgg caaaggtgga gaagggaaag tcaaagaagc tcaagtctgt caaggagctc 3540

ctgggtatca ccattatgga gaggtccagc ttcgaaaaga atccgatcga ttttctcgag 3600

gcgaagggat ataaggaagt gaagaaggac ctgatcatta agcttccaaa gtacagtctt 3660

ttcgagttgg aaaacggcag gaagcgcatg ttggcttccg caggagagct ccagaagggt 3720

aacgagcttg ctttgccgtc caagtatgtg aacttcctct atctggcatc ccactacgag 3780

aagctcaagg gcagcccaga ggataacgaa cagaagcaac tgtttgtgga gcaacacaag 3840

cattatcttg acgagatcat tgaacagatt tcggagttca gtaagcgcgt catcctcgcc 3900

gacgcgaatt tggataaggt tctctcagcc tacaacaagc accgggacaa gcctatcaga 3960

gagcaggcgg aaaatatcat tcatctcttc accctgacaa accttggggc tcccgctgca 4020

ttcaagtatt ttgacactac gattgatcgg aagagataca cttctacgaa ggaggtgctg 4080

gatgcaaccc ttatccacca atcgattact ggcctctacg agacgcggat cgacttgagt 4140

cagctcgggg gggataagag accagcggca accaagaagg caggacaagc gaagaagaag 4200

aagtag 4206

<210> 6

<211> 728

<212> DNA

<213>It is artificial synthesized

<400> 6

ggtacgctga aatcaccagt ctctctctac aaatctatct ctctctattt tctccataaa 60

taatgtgtga gtagtttccc gataagggaa attagggttc ttatagggtt tcgctcatgt 120

gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt ctatcaataa 180

aatttctaat tcctaaaacc aaaatccagt actaaaatcc agatctccta aagtccctat 240

agatctttgt cgtgaatata aaccagacac gagacgacta aacctggagc ccagacgccg 300

ttcgaagcta gaagtaccgc ttaggcagga ggccgttagg gaaaagatgc taaggcaggg 360

ttggttacgt tgactccccc gtaggtttgg tttaaatatg atgaagtgga cggaaggaag 420

gaggaagaca aggaaggata aggttgcagg ccctgtgcaa ggtaagaaga tggaaatttg 480

atagaggtac gctactatac ttatactata cgctaaggga atgcttgtat ttatacccta 540

taccccctaa taacccctta tcaatttaag aaataatccg cataagcccc cgcttaaaaa 600

ttggtatcag agccatgaat aggtctatga ccaaaactca agaggataaa acctcaccaa 660

aatacgaaag agttcttaac tctaaagata aaagatcttt caagatcaaa actagttccc 720

tcacaccg 728

<210> 7

<211> 1988

<212> DNA

<213>Corn（Zea mays）

<400> 7

gtgcagcgtg acccggtcgt gcccctctct agagataatg agcattgcat gtctaagtta 60

taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt atctatcttt 120

atacatatat ttaaacttta ctctacgaat aatataatct atagtactac aataatatca 180

gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca attgagtatt 240

ttgacaacag gactctacag ttttatcttt ttagtgtgca tgtgttctcc tttttttttg 300

caaatagctt cacctatata atacttcatc cattttatta gtacatccat ttagggttta 360

gggttaatgg tttttataga ctaatttttt tagtacatct attttattct attttagcct 420

ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaataattt agatataaaa 480

tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt aaaaaaacta 540

aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc gtcgacgagt 600

ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa gcagacggca 660

cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc gttggacttg 720

ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc ggcacggcag 780

gcggcctcct cctcctctca cggcaccggc agctacgggg gattcctttc ccaccgctcc 840

ttcgctttcc cttcctcgcc cgccgtaata aatagacacc ccctccacac cctctttccc 900

caacctcgtg ttgttcggag cgcacacaca cacaaccaga tctcccccaa atccacccgt 960

cggcacctcc gcttcaaggt acgccgctcg tcctcccccc ccccccctct ctaccttctc 1020

tagatcggcg ttccggtcca tggttagggc ccggtagttc tacttctgtt catgtttgtg 1080

ttagatccgt gtttgtgtta gatccgtgct gctagcgttc gtacacggat gcgacctgta 1140

cgtcagacac gttctgattg ctaacttgcc agtgtttctc tttggggaat cctgggatgg 1200

ctctagccgt tccgcagacg ggatcgattt catgattttt tttgtttcgt tgcatagggt 1260

ttggtttgcc cttttccttt atttcaatat atgccgtgca cttgtttgtc gggtcatctt 1320

ttcatgcttt tttttgtctt ggttgtgatg atgtggtctg gttgggcggt cgttctagat 1380

cggagtagaa ttctgtttca aactacctgg tggatttatt aattttggat ctgtatgtgt 1440

gtgccataca tattcatagt tacgaattga agatgatgga tggaaatatc gatctaggat 1500

aggtatacat gttgatgcgg gttttactga tgcatataca gagatgcttt ttgttcgctt 1560

ggttgtgatg atgtggtgtg gttgggcggt cgttcattcg ttctagatcg gagtagaata 1620

ctgtttcaaa ctacctggtg tatttattaa ttttggaact gtatgtgtgt gtcatacatc 1680

ttcatagtta cgagtttaag atggatggaa atatcgatct aggataggta tacatgttga 1740

tgtgggtttt actgatgcat atacatgatg gcatatgcag catctattca tatgctctaa 1800

ccttgagtac ctatctatta taataaacaa gtatgtttta taattatttt gatcttgata 1860

tacttggatg atggcatatg cagcagctat atgtggattt ttttagccct gccttcatac 1920

gctatttatt tgcttggtac tgtttctttt gtcgatgctc accctgttgt ttggtgttac 1980

ttctgcag 1988

<210> 8

<211> 32

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 8

tggcgctggt cttcgccgtc atgatcatcg tc 32

<210> 9

<211> 27

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 9

tacgatgagc gccaccttgc ccgggaa 27

<210> 10

<211> 524

<212> DNA

<213>Wheat（Triticum aestivuml）

<400> 10

catgaatcca aaccacacgg agttcaaatt cccacagatt aaggctcgtc cgtcgcacaa 60

ggtaatgtgt gaatattata cctgtcgtgc aaaattgcct ggcctgcaca attgctgtta 120

tagttggcgg cagggagagt tttaacattg actagcgtgc tgataatttg tgagaaataa 180

taattgacaa gtggatactg acatttgaga agagcttctg aactgttatt agtaacaaaa 240

atggaaagct gatgcacgga aaaaggaaag aaaaagccat actttttttt aggtaggaaa 300

agaaaaagcc atacgagact gatgtctctc agatgggccg ggatctgtct atctagcagg 360

cagcagccca ccaacctcac gggccagcaa ttacgagtcc ttctaaaagc tcccgccgag 420

gggcgctggc gctgctgtgc agcagcacgc ctaacattag tcccacctcg ccagtttaca 480

gggagcagaa ccagcttata agccgaggcg catcctgagg tagc 524

<210> 11

<211> 437

<212> DNA

<213>Paddy rice（Oryza sativa）

<400> 11

agtaattcat ccaggtctcc aagttctagg attttcagaa ctgcaactta ttttatcaag 60

gaatctttaa acatacgaac agatcactta aagttcttct gaagcaactt aaagttatca 120

ggcatgcatg gatcttggag gaatcagatg tgcagtcagg gaccatagca caagacaggc 180

gtcttctact ggtgctacca gcaaatgctg gaagccggga acactgggta cgttggaaac 240

cacgtgatgt gaagaagtaa gataaactgt aggagaaaag catttcgtag tgggccatga 300

agcctttcag gacatgtatt gcagtatggg ccggcccatt acgcaattgg acgacaacaa 360

agactagtat tagtaccacc tcggctatcc acatagatca aagctgattt aaaagagttg 420

tgcagatgat ccgtggc 437

<210> 12

<211> 333

<212> DNA

<213>Paddy rice（Oryza sativa）

<400> 12

tgcaagaacg aactaagccg gacaaaaaaa aaaaggagca catatacaaa ccggttttat 60

tcatgaatgg tcacgatgga tgatggggct cagacttgag ctacgaggcc gcaggcgaga 120

gaagcctagt gtgctctctg cttgtttggg ccgtaacgga ggatacggcc gacgagcgtg 180

tactaccgcg cgggatgccg ctgggcgctg cgggggccgt tggatgggga tcggtgggtc 240

gcgggagcgt tgaggggaga caggtttagt accacctcgc ctaccgaaca atgaagaacc 300

caccttataa ccccgcgcgc tgccgcttgt gtt 333

<210> 13

<211> 20

<212> DNA

<213>Wheat（Triticum aestivuml）

<400> 13

ggtgcccgct ggcggcgacg 20

<210> 14

<211> 31

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 14

ttcggggttt tgcatgtcag ctagtacgga g 31

<210> 15

<211> 18

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 15

gtggacacga accgctgc 18

<210> 16

<211> 27

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 16

gacgctgtga tgatcaatgg tgccgtg 27

<210> 17

<211> 27

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 17

agcgcgtccg tgaagtgctc ctggttc 27

<210> 18

<211> 28

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 18

gacgctgtga tgaccaatgg tgccatac 28

<210> 19

<211> 19

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 19

cgacgtgtcg gccagcgca 19

<210> 20

<211> 93

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 20

gtttcagagc tatgctggaa acagcatagc aagttgaaat aaggctagtc cgttatcaac 60

ttgaaaaagt ggcaccgagt cggtgctttt ttt 93

<210> 21

<211> 76

<212> DNA

<213>Paddy rice（Oryza sativa）

<400> 21

acaaagcacc agtggtctag tggtagaata gtaccctgcc acggtacaga cccgggttcg 60

attcccggct ggtgca 76

Claims (10)

1. a kind of carrier for genome editor, the carrier contains a sgRNA4TmC+5 structure and a Cas9 gene, It is characterized in that the nucleotide sequence of the sgRNA4TmC+5 structures such as SEQ ID NO:Shown in 20.

2. carrier according to claim 1, wherein also operability is connected with one before described sgRNA4TmC+5 structures Individual promoter, the promoter is selected from one of group being made up of promoters such as TaU3p, OsU3p, TaU6 or OsU6bp.

3. carrier according to claim 1 or 2, wherein the nucleotide sequence of described Cas9 genes such as SEQ ID NO:5 It is shown.

4. carrier according to claim 3, wherein operability is connected with one and opened before described Cas9 genes Mover, the promoter is selected from one of group being made up of Ubi or 2*35S promoters.

5. a kind of genome edit methods, methods described is completed by CRISPR-Cas9 systems, it is characterised in that described Contain a sgRNA4TmC+5 structure and a Cas9 gene, the sgRNA4TmC+5 in the carrier of CRISPR-Cas9 systems The nucleotide sequence of structure such as SEQ ID NO:Shown in 20.

6. method according to claim 5, wherein also operability is connected with one before described sgRNA4TmC+5 structures Individual promoter, the promoter is selected from one of group being made up of promoters such as TaU3p, OsU3p, TaU6 or OsU6bp.

7. the method according to claim 5 or 6, wherein the nucleotide sequence of described Cas9 genes such as SEQ ID NO:5 It is shown.

8. method according to claim 7, wherein operability is connected with one and opened before described Cas9 genes Mover, the promoter is selected from one of group being made up of Ubi or 2*35S promoters.

9. method according to claim 8, operability is gone back before sgRNA4TmC+5 structures is connected with a target base Because of sequence, the nucleotide sequence such as SEQ ID NO of the target-gene sequence:2 or SEQ ID NO:Shown in 13.

10. claim 1-4 any described carrier, claim 5-8 any described genome edit methods are being planted Application in thing genome editor.