CN104450745A - Method for acquiring specific rice gene mutant and application thereof - Google Patents

Abstract

The invention provides a method for acquiring specific rice gene mutants. Through optimization of a traditional CRISPR / Cas system, an optimized CAS9 gene expression vector, an optimized sgRNA expression vector and an optimized binary expression vector of crRNA:tracrRNA are constructed; the recognition sequences of the screened specific rice genes are cloned to the vectors to construct transformation vectors, so as to finally obtain the specific rice gene mutants. The method applies the CRISPR / Cas system to rice to generate the specific gene mutants, and has high mutation rate and wide application value in plant gene engineering.

Description

A kind of method and application thereof obtaining paddy rice appointment gene mutation body

Technical field

The invention belongs to field of plant genetic, be specifically related to a kind of in paddy rice, obtain appointment gene mutation body method and application thereof.

Background technology

Paddy rice (Oryza sativaL.) is important food crop, has supported half earth population, and Study On Rice, the grain security improving rice yield national to guarantee have important meaning.Thus to the research of paddy gene function with illustrate, and utilize existing gene function information to handle and transform paddy gene function, for improving economical character, improving grain yield and there is important Theory and applications being worth.Paddy rice is monocotyledonous model plant, has long research history; Meanwhile, other grass relatively, genome is less, is one of plant completing gene order-checking the earliest.After Sequencing of Rice Genome completes, greatly facilitate the research of paddy gene function.In forward genetics, such as, utilize the technology of map based cloning can determine to cause the gene locus of specific mutant phenotype.In reverse genetics, the T-DNA insertion mutation body of a large amount of known boundaries sequence is that the function studying specific gene offers convenience.However, these methods itself are all the principles based on randomness at present, and investigator always can not obtain the desirable mutant material of specific site.Simultaneously, the overwhelming majority still rests in the aspect of random disruptions gene function to the operation of rice genome, cannot carry out meticulousr and sequence change accurately, the sequence such as only in indivedual site to certain gene is replaced, instead of destroys the function of this gene completely; Or utilize homologous recombination to carry out the site-directed integration of reporter gene.Even if having to be reported in paddy rice and enter genomic plant by using the method for positive-negative selection mark to obtain site-directed integration simultaneously, be also that the screening amount by improving transfer-gen plant realizes, the efficiency of this strategy is very low, is not suitable for extensive employing.Also similar problem is there is for Agricultural biotechnologies, investigator needs by the generation generation hybridization repeatedly work of time and effort consuming such as to backcross, excellent proterties to be converged in the middle of new kind, thus raises the efficiency in the urgent need to method that is new, that directly can change genome sequence.

In order to realize efficient genome editor, crucial prerequisite can fix a point to produce DNA double chain break.The technology that can be used for realizing fixing a point to cut in genomic level comparatively conventional at present has the meganuclease based on Homing endonucleases (homing nuclease), based on the restriction endonuclease (Zfn) of zinc fingers, and based on the TALEN technology of Tal effector.Because Homing endonucleases can identify about 20 bases, specificity is far superior to common restriction enzyme, but the DNA differential threshold of this enzyme and cutting territory are positioned at same structure territory, thus the DNA recognition sequence of people's for a change this enzyme often causes cutting efficiency to reduce simultaneously, thus the design cost of meganuclease technology is very high, and the method that there is no at present is promoted.Based on the restriction endonuclease (Zfn) of zinc fingers, above-mentioned problem is there is not with the TALEN technology based on Tal effector, because DNA differential threshold and cutting territory separate, investigator only needs to be absorbed in design can the protein sequence of specific combination appointment DNA sequence dna.Their similarity is also, it is all the domain module identifying a few base or a base, carry out tandem sequence repeats to realize the specific recognition to specifying DNA sequence dna, this just means that running into different DNA sequence dnas all will rebuild carrier, but the tumor-necrosis factor glycoproteins of disparate modules is carried out the cloning process spliced for traditional challenge.Neoteric based on CRISPR(Clustered Regularly Interspaced Small Palindromic Repeats recently)/Cas(CRISPR associatatedsystem) the genome edit methods of system shows larger potentiality.CRISPR/Cas system finds it is a set of adaptive immune system for exogenous DNA molecule be extensively present in prokaryotic organism recently, is responsible for resisting phage invasion, the plasmid of Degradation and Transformation in eubacterium and ancient bacterium.This system can by between regular for the partial sequence of the DNA of the invasion CRISPR tumor-necrosis factor glycoproteins being incorporated into existence in advance after cell is attacked for the first time, be called introns (spacer), until when cell is again under attack, this section of sequence is together transcribed into microRNA---crRNA(CRISPRRNA).For CRISPR II type system, also exist a microRNA---tracrRNA and crRNA forms dimer, participation crRNA processing, and with crRNA together functionating.Simultaneously compared with other types, the structure of CRISPR II type system is the compactest: only need a proteolytic enzyme CAS9, just can under the guidance of crRNA:tracrRNA double base molecule, form R-loop in the DNA double chain of being invaded by spacer sequence, sequence-specific cutting is carried out to DNA in two enzymes of CAS9 site alive subsequently.In order to correct identification DNA molecular, and spacer sequence downstream (5 '-3 ' direction) there is PAM(Protospacer AdjacentMotif) motif 5 '-NGG-3 ', cleavage site is usually located at the position of 3-4 the Nucleotide in PAM upstream.The conveniently structure of expression vector, crRNA:tracrRNA double base molecule is fused to chimeric sgRNA(single guide RNA further), its 5 ' end 20nt is the region being responsible for identifying specific gene.As long as express CAS9 albumen in nucleus and sgRNA just can realize genomic dna editor so simultaneously.It take protein as the technology of core that this technology based on CRISPR/Cas system is different from above-mentioned, CRISPR/Cas system is carried out special cutting to DNA and is mediated by microRNA, for different target genes, only need to design different microRNAs.There are some researches show and be responsible for identifying that the 20nt of specific gene can be divided into two parts, 5 ' end 8nt is due to away from the PAM sequence that will identify, very little to specificity contribution, and the PAM of 5 '-NAG-3 ' type also can by the identification of a top degree simultaneously.Thus in order to realize the specificity of cutting, identify in the 20nt of specific gene, the 12nt closing on PAM needs specificity high as far as possible, uses the PAM of 5 '-NAG-3 ' type whether can find the gene locus be identified in genome if want to consider simultaneously.

Verified in various modes animal and mode trickle biology, this system is carried out transformation and optimization expression can realize genomic editor, the result delivered at present comprises the clone of people, mouse, zebra fish, fruit bat, nematode and yeast, in some of them experiment, the efficiency of sudden change even can reach 100%.

Summary of the invention

The invention provides a kind of method obtaining paddy rice appointment gene mutation body.CRISPR/Cas system after optimizing is applied to the mutant producing in paddy rice and specify gene, and the CRISPR/Cas system stability after optimization, mutation efficiency is high, and the method has a wide range of applications in plant genetic engineering.

The invention provides a kind of CRISPR/Cas system, comprise the CAS9 gene after optimization and the sgRNA after optimizing, wherein:

The nucleotide sequence of the CAS9 gene after described optimization is as shown in the SEQ ID No:1 in sequence table;

Above-mentioned optimization is for the expression of CAS9 albumen in paddy rice, to the nucleotide sequence deriving from streptococcus pyogenes (Streptococcuspyogenes), carries out codon optimized;

SgRNA(single guide RNA after described optimization) nucleotide sequence as shown in the SEQ ID No:2 in sequence table;

Above-mentioned optimization is for the expression of sgRNA in paddy rice, has carried out structure optimizing stability to the sgRNA nucleotide sequence deriving from streptococcus pyogenes (Streptococcus pyogenes).

Further, the CAS9 gene after described optimization, has merged nuclear localization signal at 5 ' end.

Further, the sgRNA after described optimization has neck ring structure, and described neck ring structure contains 13 pairing Nucleotide, in conjunction with CAS9 gene, see accompanying drawing 2(1).

Further, above-mentioned CRISPR/Cas system also comprises the double base crRNA:tracrRNA after optimization, see accompanying drawing 2(2).

The nucleotide sequence of the double base crRNA:tracrRNA after described optimization, as shown in SEQ ID No:3, the SEQ IDNo:4 in sequence table, wherein, is the nucleotide sequence of the crRNA after optimizing shown in SEQ ID No:3; It is the nucleotide sequence of the tracrRNA after optimizing shown in SEQ ID No:4;

Above-mentioned optimization is for the expression of double base crRNA:tracrRNA in paddy rice, has carried out structure optimizing stability to the double base crRNA:tracrRNA nucleotide sequence deriving from streptococcus pyogenes (Streptococcus pyogenes)..

The invention provides a kind of expression vector, the expression regulation sequence comprising above-mentioned nucleotide sequence and be connected with above-mentioned nucleotide sequence operability.

Further, described expression regulation sequence comprises the regulating and controlling sequence of composing type high expression level, the promotor of the corn UBIQUITIN gene such as driving CAS9 to express, and the promotor of the U3 snoRNA gene driving guide RNA (sgRNA or double base crRNA:tracrRNA) to express.

Further, the expression vector of the sgRNA after optimization provided by the invention, can insert multiple sgRNA and multiple gene.

The invention provides the recognition sequence of the specific gene for paddy rice full-length genome designed, and provide in the expression vector of the sgRNA after by these sequence clones to optimization or double base crRNA:tracrRNA, build the method for conversion carrier.

Present invention also offers and utilize the conversion carrier built to obtain the method for specifying paddy gene mutant, comprise the following steps:

1) expression vector of the sgRNA after the expression vector of the CAS9 gene after optimizing, optimization or double base crRNA:tracrRNA is obtained;

2) screen the recognition sequence of specific gene and it be cloned into respectively in the expression vector of the sgRNA after optimization or double base crRNA:tracrRNA;

3) by step 2) clone in the expression vector that obtains with optimize after the expression vector of CAS9 gene be assembled into whole carrier;

4) by above-mentioned whole vector introduction rice cell, obtain paddy rice and specify gene mutation body.

Further, the recognition sequence screening specific gene comprises specificity screening and structural stability screening.

Further, the recognition sequence screening specific gene comprises the following steps:

I) select the 23nt sequence ended up with 5 '-NGG-3 ', comprise the 3nt of 20nt and PAM of spacer, the sequence comprising continuous five T is got rid of;

15 bases of ii) holding 3 ', comprise the 3nt of 12nt and PAM of spacer, carry out the comparison of BLAST or BOWTIE, get rid of not specified sequence;

Iii) the 20ntspacer sequence chosen and sgRNA sequence are merged, carry out the prediction of RNA secondary structure with RNAfold server.

Further, step I) in, described N is A or T or C or G.

Present invention also offers above-mentioned acquisition and specify the application of the method for paddy gene mutant in editor's rice genome.

Beneficial effect of the present invention is:

A) in CRISPR/Cas system provided by the invention, carrier is divided into two portions, CAS9 part and guide RNA (sgRNA or double base crRNA:tracrRNA) part.These two portions can be combined into an independent carrier by LR reaction, wherein on the carrier of guide RNA, can select to place multiple sgRNA or place multiple spacer in a double base crRNA:tracrRNA, can reach simultaneously for the object of multiple gene;

B) position (i.e. neck ring structure) of sgRNA and CAS9 combination provided by the invention is longer, and containing 13 pairing Nucleotide, RNA secondary structure is more stable, and efficiency is higher;

C) recognition sequence (spacer) that uses in the present invention, is all through RNA secondary structure and folds and analyzed, thus can ensure the stability of neck ring structure and the high specific of recognition sequence;

D) method provided by the invention, mutation efficiency is high, can reach 50%, have a wide range of applications in plant genetic engineering.

Accompanying drawing explanation

Fig. 1 (1) shows the expression vector structure iron of the CAS9 after the present invention's optimization.

Fig. 1 (2) shows the expression vector structure iron of the sgRNA after the present invention's optimization.

Fig. 1 (3) shows the expression vector structure iron of the double base crRNA:tracrRNA after the present invention's optimization.

Fig. 2 (1) shows the secondary structure schematic diagram of the sgRNA of the spacer comprised for LAZY1 gene.

Fig. 2 (2) shows the secondary structure schematic diagram of the double base crRNA:tracrRNA of the spacer comprised for CAO1 gene.

Fig. 3 (1) shows the embodiment of the present invention with the folding secondary structure figure of the sgRNA of the recognition sequence (spacer) of LAZY1 gene and double base crRNA:tracrRNA.Dotted line frame table understands in neck ring structure the position that can form A-U/G-C pairing.

Fig. 3 (2) shows the embodiment of the present invention with the folding secondary structure figure of the sgRNA of the recognition sequence (spacer) of CAO1 gene and double base crRNA:tracrRNA.Dotted line frame table understands in neck ring structure the position that can form A-U/G-C pairing.

The recognition site that Fig. 4 (1) shows embodiment of the present invention LAZY1 gene is relative to the mutational site of the LAZY1 gene mutation body obtained in the position of LAZY1 gene and embodiment 5, wherein, the sequence of PAM is represented by underscore, and spacer sequence is for highlighting part; The position (disappearance and replacement) of sudden change is for shown in square frame.

The recognition site that Fig. 4 (2) shows CAO1 gene is relative to the mutational site of the CAO1 gene mutation body obtained in the position of CAO1 gene and embodiment 5, and wherein, the sequence of PAM is represented by underscore, and spacer sequence is for highlighting part; The position (disappearance and replacement) of sudden change is for shown in square frame.

Fig. 4 (3) shows the mutational site of the LAZY1 gene mutation body obtained in the embodiment of the present invention 5, and wherein, the sequence of PAM is represented by underscore, and spacer sequence is for highlighting part.

Fig. 4 (4) shows the mutational site of the LAZY1 gene mutation body obtained in the embodiment of the present invention 5, and wherein, the sequence of PPAM is represented by underscore, and spacer sequence is for highlighting part.

Embodiment

Embodiment 1, in rice cell core high level expression CAS9 carrier and in nucleus, express the structure of carrier of microRNA.

First the CAS9 gene order of Streptococcus pyogenes has merged nuclear localization signal at 5 ' end, and then codon optimized (SEQ ID No:1) has been carried out for the genetic expression of paddy rice in whole coding region.Then with Ji Busen splicing method the constitutive promoter Ubi from corn, CAS9, be cascaded with 35S terminator and be cloned into conversion carrier pH2GW7(purchased from Ghent, Belgium university http://gateway.psb.ugent.be/) in, substituted for the 35S promoter that this carrier carries simultaneously.The carrier called after pH-Ubi-CAS9-7 checking order correct, as shown in Fig. 1 (1).

The sgRNA gene order of Streptococcus pyogenes simulates the secondary structure of double base crRNA:tracrRNA to greatest extent, as shown in Fig. 3 (1), Fig. 3 (2).Add two BsaI or two BsmBI restriction enzyme sites and 8 continuous print Nucleotide T by after japonica rice U3snRNA promoter sequence, as terminator, obtain Entry carrier by Topo clone.By two sgRNA(SEQ ID No:2) or double base crRNA:tracrRNA(SEQ ID No:3, SEQ ID No:4) be cloned into BsaI and BsmBI inside respectively, as shown in Fig. 4 (1), Fig. 4 (2), the carrier called after pOS-sgRNA(SEQ ID No:18 finally obtained) and pOS-cr-tracr(SEQ ID No:19, SEQ ID No:20), respectively as shown in Fig. 1 (2), Fig. 1 (3).Two BsaI or two BsmBI restriction enzyme site is still remained for cloning the recognition sequence of the specific gene of embodiment 2 screening in pOS-sgRNA and pOS-crRNA.

The recognition sequence (spacer) of embodiment 2, screening specific gene.

The transcript sequence of each gene obtains from MSU/TIGR rice genome database ( http:// rice.plantbiology.msu.edu/).The first step selects the 23nt sequence (3nt of 20nt and PAM of spacer) ended up with 5 '-NGG-3 ', and N is A/T/C/G any one here.The sequence comprising continuous five T is got rid of.Second step, 15 bases of holding 3 ', comprise the 3nt of 12nt and PAM of spacer, carry out the comparison of BLAST or BOWTIE, get rid of not specified sequence.Repeat the non-specific activity that second step can get rid of 5 '-NAG-3 ' PAM further.The specific recognition sequence comprising whole paddy gene can arrive ftp.cbi.pku.edu.cn/pub/supplementary_file/ and download.According to this list, we have chosen the specific recognition sequence for CAO1 and LAZY1 gene.3rd step, merges the 20nt spacer sequence chosen and sgRNA sequence, carries out the prediction of RNA secondary structure with RNAfold server.We have 2 points to secondary structure accepted standard, and one is stable neck ring structure, and two is as far as possible unpaired spacer districts.

Particularly, when spacer and sgRNA other sequence not very by force in conjunction with, the normal folding of other sequence of sgRNA can not be affected on the one hand, ensure that the position (neck ring structure) that sgRNA and CAS9 combines is very stable, prevent other sequence of spacer and sgRNA combine and can not insert smoothly in target DNA double-strand on the other hand.

The eliminating of distinguished sequence is not the important factor improving final mutation efficiency yet.

Embodiment 3, clone for the specific recognition sequence of CAO1 and LAZY1 gene.

Add 5 '-GGCA-3 ' at the 5 ' end of the spacer chosen, add 5 '-AAAC-3 ', the specific recognition sequence for for CAO1 and LAZY1 gene at the complementary strand 5 ' end of the spacer chosen:

cao1-f1 5’-GGCAaccggaccacctcaagagct-3’（SEQ ID No：5）

cao1-r1 5’-AAACagctcttgaggtggtccggt-3’（SEQ ID No：6）

lazy-f1 5’-GGCAgcgtcggcggagacagagcg-3’（SEQ ID No：7）

lazy-r1 5’-AAACcgctctgtctccgccgacgc-3’（SEQ ID No：8）

After synthetic primer, annealing forms BsaI or BsmBI restriction enzyme site in carrier pOS-sgRNA that namely double-strand can be cloned into sgRNA.Add 5 '-GGCA-3 ' at the 5 ' end of the spacer chosen, add 5 '-TAAAAC-3 ', the specific recognition sequence for for CAO1 and LAZY1 gene at the complementary strand 5 ' end of the spacer chosen:

cao1-f2 5’-GGCAtggatatcgaaccggaccacctcaagagct-3’（SEQ ID No：9）

cao1-r2 5’-TAAAACagctcttgaggtggtccggttcgatatcca-3’（SEQ ID No：10）

lazy-f2 5’-GGCAgctgctgccggcgtcggcggagacagagcg-3’（SEQ ID No：11）

lazy-r2 5’-TAAAACcgctctgtctccgccgacgccggcagcagc-3’（SEQ ID No：12）

After synthetic primer, annealing forms BsaI restriction enzyme site in carrier pOS-cr-tracr that namely double-strand can be cloned into double base crRNA:tracrRNA.The Entry carrier of having cloned enters pH-Ubi-CAS9-7 carrier by LR reaction, obtains being used for the whole carrier of rice transformation.

The condition of above-mentioned LR reaction is the system of 5 μ L, the LR enzyme (Invitrogen) of the pH-Ubi-CAS9-7 containing 1 μ L, pOS-sgRNA or pOS-cr-tracr of 1 μ L, 1 μ L, the H of 2 μ L ₂o.In 22 DEG C of reactions 1 hour.

Embodiment 4, transform precocious japonica rice variety Kitaake

Rice callus is induced:

Choose full Kitaake rice paddy seed (from crop investigations institute of the Chinese Academy of Agricultural Sciences), peel off seed coat, after sterilizing washing, click and enter at the sterilizing NB solid medium with 2 mg/litre 2,4-D uniformly, 32 DEG C of continuous lights, 5 days callus inductions are formed.

Agrobacterium-mediated Transformation:

Simultaneously by pH-Ubi-CAS9-7 carrier heat shock method transformation Agrobacterium EHA105 competent cell (the Hood EE with spacer, Gelvin SB, Melchers S, Hoekema A (1993) New Agrobacterium helper plasmids forgene transfer to plants (EHA105) .Trans Res2:208-218), be coated with the solid LB media with the grand element of 50 micrograms per litre, 28 DEG C of dark culturing, after 2 days, obtain positive colony.The positive colony obtained in the solid AB substratum 28 DEG C of dark culturing 3 days containing 50 mg/litre kantlex, for rice conversion.

Rice callus transforms:

Washed down by Agrobacterium from AB substratum with the liquid conversion substratum containing 100 micromoles per liter Syringylethanones of filtration sterilization, bacterial concentration is adjusted to OD600 about 0.08.Choose the callus that upgrowth situation is good, soak 2 minutes in bacterium liquid, dry on aseptic filter paper afterwards, then callus is moved to the NB Dual culture substratum containing 100 micromoles per liter Syringylethanones, Dual culture 3 days under 25 DEG C of dark.

Screening of Rice callus:

Dual culture is after 3 days, callus is washed 5 times with sterilized water, again with the aseptic washing one time containing 500 mg/litre carbenicillin disodiums of 200 milliliters, carefully remove liquid, with aseptic nipper by callus gripping on aseptic filter paper, NB screening culture medium (the damp enzyme element of 2,4-D, 50 mg/litre containing 2 mg/litre and the carbenicillin disodium of 400 mg/litre) is transferred to, 32 DEG C of continuous lights 2 weeks after drying.

The differentiation of positive callus:

Choose the positive callus that well-grown is bright yellow, move to the pre-division culture medium of NB (the damp enzyme element containing 1 mg/litre NAA, 5 mg/litre ABA, 2 mg/litre kinetin, 25 mg/litre and the carbenicillin disodium of 200 mg/litre) with aseptic nipper, 32 DEG C of continuous lights are cultivated.Select eugonic callus after 2 weeks and proceed to MS division culture medium (the damp enzyme element containing 0.02 mg/litre NAA, 2 mg/litre kinetin, 50 mg/litre and the carbenicillin disodium of 200 mg/litre), 32 DEG C of continuous lights are cultivated.Wait that the seedling breaking up out grows to 2 to 5 millimeters, proceed to not containing hormone and antibiotic MS culture medium culturing 2 to 3 week, move in soil afterwards and be placed in greenhouse-grown (temperature 28-30 DEG C, 16 h light/8 h dark).

Embodiment 5, T1 to be identified for the genotype of CAO1 and LAZY1 in transgenic paddy rice.

The DNA of transfer-gen plant is extracted by CTAB method.PCR primer according to the DNA sequence dna design gene specific of CAO1 gene: CF, 5 '-CAC CGC TTT CCC CCA GTA ACC ACA-3 ' (SEQ ID No:13); CR, 5 '-TCCGAA ACT TCA ACC TCA AGC-3 ' (SEQ ID No:14).PCR primer according to the DNA sequence dna design gene specific of LAZY1 gene: LF, 5 '-CCT GAC CAC GAC AAC GAC TAC TTC-3 ' (SEQ ID No:15); LR, 5 '-ACA CCA AGG TGT GCA GTG CTA CTC-3 ' (SEQ ID No:16).

The present invention, by rice transformation callus, obtains the transgenic paddy rice containing CRISPR/Cas system, finds that the effect render transgenic paddy rice CAO1 gene of CRISPR/Cas system and LAZY1 gene locus there occurs sudden change by genotype identification and order-checking.. for LAZY1 gene, we find 6 strains to be that two karyomit(e)s all there occurs consistent sequence deletion from 12 strains independently transgenic plant, mutation efficiency is high, can 50% be reached, and the mutated site of this six strain is different from each other, to lack the mutant strain of 2bp, 760bp and 94bp base respectively, the mutational site of the LAZY1 gene mutation body of acquisition is as shown in Fig. 4 (1), (3), (4); For CAO1 gene, the mutational site of the CAO1 gene mutation body obtained is as shown in Fig. 4 (2), and wherein, the sequence obtained after sudden change is represented by black matrix, the sequence of disappearance is represented by strigula, and the 92bp sequence that CAO1 gene mutation body obtains is as shown in sequence table SEQ ID No:17.As shown in Fig. 4 (1)-(4), mutational site all round the DNA double chain cleavage site of theoretical prediction, indicates the reliability of result.

Above method specifies the rice mutant of gene significant for obtaining with transgenic method.

Claims (12)

1. a CRISPR/Cas system, comprises the CAS9 gene after optimization and the sgRNA after optimizing, wherein:

The nucleotide sequence of the CAS9 gene after described optimization is as shown in the SEQ ID No:1 in sequence table;

The nucleotide sequence of the sgRNA after described optimization is as shown in the SEQ ID No:2 in sequence table.

2. CRISPR/Cas system as claimed in claim 1, it is characterized in that, the sgRNA after described optimization has neck ring structure, and described neck ring structure contains 13 pairing Nucleotide, in conjunction with CAS9 gene.

3. CRISPR/Cas system as claimed in claim 1, is characterized in that, also comprise the double base crRNA:tracrRNA after optimization.

4. CRISPR/Cas system as claimed in claim 3, it is characterized in that, the nucleotide sequence of the double base crRNA:tracrRNA after described optimization is as shown in the SEQ ID No:3 in sequence table, SEQ ID No:4, wherein, shown in SEQ ID No:3 be the nucleotide sequence of crRNA after optimizing; It is the nucleotide sequence of the tracrRNA after optimizing shown in SEQ ID No:4.

5. expression vector, the nucleotide sequence in the CRISPR/Cas system described in claim 1-4 is arbitrary and the expression regulation sequence be connected with above-mentioned nucleotide sequence operability are formed.

6. expression vector as claimed in claim 5, it is characterized in that, described expression regulation sequence comprises the regulating and controlling sequence of composing type high expression level.

7. expression vector as claimed in claim 5, it is characterized in that, described expression vector, comprise the expression vector of the expression vector of the sgRNA after the expression vector of the CAS9 gene after optimization, optimization and the double base crRNA:tracrRNA after optimizing, the expression vector sequence of the sgRNA after wherein optimizing is as shown in sequence table SEQ ID No:18, and the expression vector of the double base crRNA:tracrRNA after optimization is as shown in sequence table SEQ ID No:19, SEQ ID No:20.

8. obtain a method of specifying paddy gene mutant, comprise the following steps:

1) expression vector according to claim 7 is obtained;

2) screen the recognition sequence of specific gene and it be cloned into respectively in the expression vector of the sgRNA after optimization or double base crRNA:tracrRNA;

3) by step 2) clone in the expression vector that obtains with optimize after the expression vector of CAS9 gene be assembled into whole carrier;

4) by above-mentioned whole vector introduction rice cell, obtain paddy rice and specify gene mutation body.

9. method as claimed in claim 8, is characterized in that, the recognition sequence of described screening specific gene comprises specificity screening and the structural stability screening of recognition sequence.

10. method as claimed in claim 8, is characterized in that, the recognition sequence of screening specific gene comprises the following steps:

I) select the 23nt sequence ended up with 5 '-NGG-3 ', comprise the 3nt of 20nt and PAM of spacer, the sequence comprising continuous five T is got rid of;

15 bases of ii) holding 3 ', comprise the 3nt of 12nt and PAM of spacer, carry out the comparison of BLAST or BOWTIE, get rid of not specified sequence;

Iii) the 20nt spacer sequence chosen and sgRNA sequence are merged, carry out the prediction of RNA secondary structure with RNAfold server.

11. methods as claimed in claim 10, is characterized in that, step I) in, described N is A or T or C or G.

The application of the arbitrary described method of 12. claim 8-11 in editor's rice genome.