CN117384948A - Gene sequence for realizing A/T accurate replacement at target site-4 position and replacement method - Google Patents

Abstract

The invention provides a gene sequence for realizing A/T accurate replacement at a target site-4 position and a replacement method. According to the invention, nmCas9 and SpCas9 double expression frames are introduced into a single vector, experiments prove that SpCas9 is utilized to relatively favor base deletion and NmCas9 relatively favor single base insertion in plants, target sgRNA which is matched with SpCas9 and sgRNA which is matched with NmCas9 and has-4 single base deletion in a target sequence are introduced into genome targets of a co-tangent point, so that a rice targeting editing vector is constructed, rice can be caused to replace A or T at any single base at-4 positions after being introduced into rice cells, and therefore, accurate single base replacement of a DSB template-free plant genome is realized.

Description

Gene sequence for realizing A/T accurate replacement at target site-4 position and replacement method

Technical Field

The invention relates to the technical fields of biotechnology and plant genetic engineering. Specifically, the invention relates to a gene sequence for realizing A/T accurate replacement at a target site-4, and a method for realizing A/T accurate replacement at the target site-4 by utilizing SpCas9 and NmCas9 double expression frames.

Background

Key to crop breeding is the creation and use of genetic variation. The genetic variation utilized in traditional crop breeding mostly comes from accumulation of natural mutation in the domestication process or artificial physicochemical mutagenesis; however, these mutations are randomly generated in the genome, and the variable resources available for improvement of agronomic traits are extremely limited, severely affecting the speed and effectiveness of variety improvement. The gene editing technology, in particular the CRISPR-Cas9 gene editing technology developed since 2013, can introduce variation at specific loci of genome, and brings 'subversion' breakthrough for crop breeding. The most widely applied crop gene editing system at present is to realize the knockout of target genes by introducing small fragment insertion or deletion at fixed points. However, many important traits of crops are often associated with a particular small number of base variations rather than gene deletions.

In order to achieve accurate editing of eukaryotic genomes, particularly high frequency base substitution, a range of HDR-independent editing tools have been developed and widely used. The single base editing technology is a precise editing technology developed based on a CRISPR-Cas system, single base controllable substitution can be introduced without DSB and donor DNA repair templates, but the single base editing system can only finish 6 types of 12 base conversion types and can only efficiently act in an editing window of about 5bp, so that the targeting capability is doubly limited by PAM and the editing window. The guide editing system is a recently developed precise gene editing technology, and can complete any type of base substitution and precise small fragment insertion and deletion; and a plurality of sites in the PAM distal end sequence can be edited efficiently, the targeting range is basically unlimited, but the efficiency of guiding editing in plants is low. Therefore, expanding the precise editing thought and developing a new precise editing strategy has important significance for realizing precise manipulation of plant genome and accelerating crop breeding improvement.

Disclosure of Invention

Aiming at the problem that the prior art can not accurately realize that any base of a specific site is replaced by A/T, the invention integrates a double expression frame of Nm1Cas9 and SpCas9 on a single plant expression vector, then introduces target sgRNA which is matched with SpCas9 and sgRNA which is matched with Nm1Cas9 and has-4 single base deletion in a target sequence aiming at a rice genome locus with a PAM sequence of NGGNGATT, realizes the base deletion at the A site through the SpCas9 expression frame, and then carries out T site insertion at the deletion position through the Nm1Cas9 expression frame, thereby realizing that the arbitrary single base at the-4 site is replaced by A or T at high frequency, and realizing the accurate single base substitution of the plant genome without a DSB template.

According to the invention, firstly, nm1Cas9 and SpCas9 expression frames are assembled respectively by using a Gibson method and are sequentially subjected to enzyme digestion and connected to the same carrier, on the basis of the carrier, a Goldengate method is adopted for a single target point to obtain a targeting carrier containing Nm1Cas9 and SpCas9, and the mutation efficiency of site-specific substitution A/T is detected through rice genetic transformation and targeted sequencing. Comprising the following steps:

(1) Removing shell of rice seeds, sterilizing, separating embryo, and placing on callus induction medium to generate secondary callus;

(2) Transferring the secondary callus to a new callus induction medium for preculture;

(3) Contacting the callus obtained in step (2) with agrobacterium carrying a targeting vector for SpCas9-Nm1Cas9 for 15 minutes;

(4) Transferring the callus of the step (3) to a culture dish on which three sterile filter papers (2.5-3.5 mL of agrobacterium suspension medium is added) are placed, and culturing at 21-23 ℃ for 48 hours;

(5) Placing the callus in the step (4) on a pre-screening culture medium for culturing for 5-7 days;

(6) Transferring the callus of step (5) onto a screening medium to obtain a resistant callus;

(7) Transferring the resistant callus to a differentiation regeneration medium to differentiate into seedlings; and is also provided with

(8) Transferring the seedlings in the step (7) into a rooting culture medium for rooting.

Wherein the seed in step (1) is a mature seed; the induction medium in the steps (1) and (2) is an induction medium listed in the explanatory table 1; contacting with agrobacterium in step (3) is immersing the callus in the agrobacterium suspension; the agrobacterium suspension medium in step (4) is the suspension medium listed in table 1 of the specification; the pre-screening medium in step (5) is illustrative of the pre-screening medium listed in table 1; the screening media of step (6) are those listed in Table 1; the differentiation and regeneration medium in the step (7) is a differentiation and regeneration medium listed in table 1 of the specification; the rooting medium in step (8) is the rooting medium listed in Table 1.

In a preferred embodiment, wherein the rice is japonica rice, more preferably, the rice is japonica Nipponbare.

Table 1 exemplary formulation of the medium

TABLE 1

In a preferred embodiment, the nucleotide sequences of the NmCas9 and SpCas9 genes are the nucleotide sequences shown in SEQ ID No.1, specifically as follows:

NM1Cas9 expression frame (promoter and terminator)

tgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaattaccacatattttttttgtcacacttg

tttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtactacaataatatcagtgttttagagaat

catataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagttttatctttttagtgtgcatgtgttctc

ctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttagggttaatggtttttatagactaattttttt

agtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataatttagatataaaatagaataaaat

aaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttcgagtagataatgccagcctgtta

aacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagcgaagcagacggcacggcatct

ctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatccagaaattgcgtggcggagcg

gcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcaccggcagctacgggggattcctttcccaccgctccttcg

ctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttcggagcgcacacacacacaa

ccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccccccctctctaccttctctagat

cggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgttagatccgtgctgctagcgttc

gtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatcctgggatggctctagccgttc

cgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaatatatgccgtgcacttgtttgtcg

ggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggagtagaattctgtttcaaactacct

ggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggatggaaatatcgatctaggatag

gtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtggtgtggttgggcggtcgttcat

tcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgtgtcatacatcttcatagttacga

gtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatacatgatggcatatgcagcatctat

tcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatatacttggatgatggcatatgcagc

agctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctcaccctgttgtttggtgttacttct

gcagGCCACCATGGCCCCAAAGAAGAAGCGCAAGGTCGCCGCGTTCAAGCCAAATT

CCATCAATTATATCCTGGGCCTGGATATCGGGATTGCGAGCGTGGGGTGGGCCATG

GTCGAGATTGATGAGGAGGAGAATCCGATCAGGCTCATTGATCTCGGCGTGAGGG

TGTTCGAGAGGGCCGAGGTCCCAAAAACCGGCGACTCCCTGGCCATGGCGAGGA

GGCTGGCGAGGTCCGTGAGGAGGCTGACCAGGAGGCGCGCGCATCGCCTGCTCA

GGACAAGGAGGCTCCTCAAGAGGGAGGGCGTGCTCCAGGCCGCCAACTTCGATG

AGAATGGGCTCATCAAGAGCCTCCCGAACACCCCGTGGCAACTCCGCGCTGCTGC

TCTCGACAGGAAGCTGACACCGCTCGAGTGGTCCGCCGTGCTCCTCCATCTGATCA

AACACCGCGGCTACCTCTCCCAACGCAAAAACGAGGGGGAAACTGCGGACAAGG

AACTGGGGGCCCTCCTGAAAGGCGTCGCCGGCAATGCGCACGCGCTCCAGACCG

GGGACTTCAGGACACCAGCCGAGCTGGCGCTCAACAAGTTCGAAAAGGAATCCG

GCCATATTAGGAACCAGCGCAGCGACTACAGCCACACCTTTAGCAGGAAGGACCT

GCAGGCGGAGCTGATCCTCCTCTTCGAGAAACAGAAGGAGTTCGGCAACCCACAT

GTCAGCGGCGGCCTCAAGGAAGGCATCGAAACACTCCTCATGACCCAACGCCCAG

CCCTGAGCGGCGATGCGGTCCAGAAGATGCTCGGCCATTGTACCTTTGAACCAGC

GGAGCCGAAGGCCGCGAAAAATACCTATACCGCCGAGCGCTTCATTTGGCTGACC

AAGCTCAACAACCTCAGGATCCTCGAACAGGGCAGCGAGAGGCCGCTGACAGAC

ACCGAACGCGCCACCCTGATGGATGAGCCGTACAGGAAGTCCAAGCTCACCTACG

CCCAGGCCCGCAAACTCCTCGGCCTGGAGGATACCGCCTTCTTCAAGGGGCTCCG

CTATGGCAAGGATAATGCCGAAGCGTCCACCCTCATGGAAATGAAGGCGTACCATG

CCATCAGCCGCGCCCTGGAGAAGGAAGGGCTCAAGGACAAGAAGAGCCCGCTGA

ATCTCAGCCCGGAACTGCAGGATGAGATCGGCACAGCCTTCTCCCTGTTCAAAAC

CGATGAGGACATTACCGGCCGCCTGAAAGACCGCATCCAACCAGAAATCCTCGAG

GCCCTCCTGAAGCACATCTCCTTTGATAAATTCGTGCAGATTAGCCTCAAGGCCCT

CAGGAGGATCGTGCCGCTCATGGAGCAAGGGAAAAGGTACGACGAGGCGTGCGC

CGAGATTTACGGCGATCACTACGGGAAGAAGAATACCGAAGAGAAGATTTACCTG

CCGCCGATTCCAGCCGACGAAATCAGGAATCCGGTCGTCCTCAGGGCCCTGTCCC

AGGCGCGCAAGGTGATTAACGGCGTGGTGAGGCGCTACGGCAGCCCGGCCAGGAT

CCACATCGAAACAGCGCGCGAAGTCGGCAAGTCCTTCAAGGACAGGAAGGAGAT

CGAGAAGAGGCAGGAAGAGAATCGCAAGGACAGGGAGAAGGCGGCGGCGAAGT

TTAGGGAATACTTCCCGAACTTTGTGGGGGAGCCAAAGAGCAAGGACATCCTGAA

GCTCAGGCTGTATGAACAACAGCACGGCAAGTGCCTGTATTCCGGCAAGGAGATC

AACCTGGGGAGGCTGAACGAGAAGGGCTATGTGGAGATCGACCATGCGCTCCCGT

TCTCCCGCACCTGGGACGACTCCTTCAACAACAAGGTGCTCGTGCTCGGGAGCGA

AAATCAGAATAAAGGGAACCAAACACCGTACGAGTACTTCAACGGCAAGGACAA

CAGCCGCGAGTGGCAGGAGTTTAAGGCGAGGGTCGAGACATCCCGCTTTCCGCGC

AGCAAGAAGCAGCGCATTCTCCTCCAGAAATTCGACGAGGATGGGTTCAAAGAGC

GCAACCTCAACGATACCAGGTACGTGAACAGGTTCCTCTGCCAATTTGTCGCGGAT

AGGATGCGCCTCACCGGCAAGGGCAAGAAGCGCGTCTTTGCGTCCAATGGGCAGA

TCACAAACCTGCTGAGGGGCTTCTGGGGCCTGAGGAAAGTGCGCGCGGAGAATG

ATAGGCATCACGCGCTGGACGCCGTGGTCGTGGCGTGCTCCACAGTCGCGATGCA

GCAAAAAATCACACGCTTCGTCCGCTACAAAGAGATGAACGCGTTCGACGGCAAA

ACCATCGACAAGGAAACTGGGGAGGTCCTGCACCAGAAGACCCACTTCCCACAA

CCGTGGGAGTTTTTCGCGCAGGAGGTCATGATCAGGGTGTTCGGCAAACCGGACG

GCAAGCCGGAGTTCGAAGAGGCGGATACCCTGGAGAAGCTCAGGACCCTGCTGG

CGGAGAAACTGTCCAGCAGGCCGGAAGCGGTGCATGAGTACGTCACCCCGCTCTT

CGTCTCCAGGGCGCCGAACAGGAAGATGAGCGGCCAAGGCCACATGGAAACAGT

GAAGTCCGCGAAACGCCTCGACGAGGGGGTGAGCGTCCTCAGGGTGCCACTCAC

CCAACTCAAACTCAAGGACCTGGAGAAGATGGTGAATCGCGAGAGGGAGCCAAA

GCTCTATGAGGCCCTGAAGGCCCGCCTGGAAGCCCACAAAGACGACCCAGCGAA

GGCCTTCGCGGAGCCGTTTTATAAGTACGACAAAGCCGGGAACAGGACCCAGCAG

GTCAAAGCCGTGAGGGTCGAACAGGTCCAGAAAACCGGGGTGTGGGTGCGCAAT

CATAACGGGATCGCCGATAATGCCACAATGGTCAGGGTGGATGTGTTCGAGAAAG

GCGATAAGTACTACCTGGTGCCGATCTACAGCTGGCAAGTCGCCAAAGGCATTCTC

CCGGATCGCGCGGTGGTGCAGGGCAAGGATGAAGAGGACTGGCAGCTGATCGAC

GATTCCTTTAATTTCAAGTTCAGCCTGCACCCGAATGACCTCGTGGAGGTCATCAC

CAAGAAAGCCCGCATGTTTGGCTACTTCGCCTCCTGTCACAGGGGCACCGGCAATA

TCAATATCCGCATCCACGACCTCGACCATAAGATCGGCAAAAACGGCATCCTGGAG

GGGATTGGCGTCAAAACAGCGCTGAGCTTCCAGAAGTACCAGATTGACGAATTAG

GCAAAGAGATCAGGCCGTGCAGGCTCAAGAAACGCCCACCGGTCAGGTACCCATA

CGATGTTCCAGATTACGCTAAGCGGCCAGCGGCGACGAAGAAGGCGGGGCAGGC

GAAGAAGAAGAAGTAAGAGCTCaagggtgggcgcgccgacccagctttcttgtacaaagtggtgatatcccgcg

gccatggcggccgggagcatgcgacgtcgatctaactgactagccgcggccatgctagagtccgcaaaaatcaccagtctctctctac

aaatctatctctctctatttttctccagaataatgtgtgagtagttcccagataagggaattagggttcttatagggtttcgctcatgtgttgag

catataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtgacctgca

SpCas9 expression cassette (promoter and terminator)

aagcttgatatcgaattcctgcagtgcagcgtgacccggtcgtgcccctctctagagataatgagcattgcatgtctaagttataaaaaatt

accacatattttttttgtcacacttgtttgaagtgcagtttatctatctttatacatatatttaaactttactctacgaataatataatctatagtacta

caataatatcagtgttttagagaatcatataaatgaacagttagacatggtctaaaggacaattgagtattttgacaacaggactctacagtt

ttatctttttagtgtgcatgtgttctcctttttttttgcaaatagcttcacctatataatacttcatccattttattagtacatccatttagggtttaggg

ttaatggtttttatagactaatttttttagtacatctattttattctattttagcctctaaattaagaaaactaaaactctattttagtttttttatttaataa

tttagatataaaatagaataaaataaagtgactaaaaattaaacaaataccctttaagaaattaaaaaaactaaggaaacatttttcttgtttc

gagtagataatgccagcctgttaaacgccgtcgacgagtctaacggacaccaaccagcgaaccagcagcgtcgcgtcgggccaagc

gaagcagacggcacggcatctctgtcgctgcctctggacccctctcgagagttccgctccaccgttggacttgctccgctgtcggcatc

cagaaatgcgtggcggagcggcagacgtgagccggcacggcaggcggcctcctcctcctctcacggcacggcagctacggggga

ttcctttcccaccgctccttcgctttcccttcctcgcccgccgtaataaatagacaccccctccacaccctctttccccaacctcgtgttgttc

ggagcgcacacacacacaaccagatctcccccaaatccacccgtcggcacctccgcttcaaggtacgccgctcgtcctccccccccc

cccctctctaccttctctagatcggcgttccggtccatggttagggcccggtagttctacttctgttcatgtttgtgttagatccgtgtttgtgtt

agatccgtgctgctagcgttcgtacacggatgcgacctgtacgtcagacacgttctgattgctaacttgccagtgtttctctttggggaatc

ctgggatggctctagccgttccgcagacgggatcgatttcatgattttttttgtttcgttgcatagggtttggtttgcccttttcctttatttcaat

atatgccgtgcacttgtttgtcgggtcatcttttcatgcttttttttgtcttggttgtgatgatgtggtctggttgggcggtcgttctagatcggag

tagaattctgtttcaaactacctggtggatttattaattttggatctgtatgtgtgtgccatacatattcatagttacgaattgaagatgatggat

ggaaatatcgatctaggataggtatacatgttgatgcgggttttactgatgcatatacagagatgctttttgttcgcttggttgtgatgatgtg

gtgtggttgggcggtcgttcattcgttctagatcggagtagaatactgtttcaaactacctggtgtatttattaattttggaactgtatgtgtgt

gtcatacatcttcatagttacgagtttaagatggatggaaatatcgatctaggataggtatacatgttgatgtgggttttactgatgcatatac

atgatggcatatgcagcatctattcatatgctctaaccttgagtacctatctattataataaacaagtatgttttataattattttgatcttgatata

cttggatgatggcatatgcagcagctatatgtggatttttttagccctgccttcatacgctatttatttgcttggtactgtttcttttgtcgatgctc

accctgttgtttggtgttacttctgcagcccgggggatccccaatacttgtatggccgcggccgcgccaccatggccccaaagaagaag

cgcaaggtcgacaagaagtactccatcggcctcgacatcggcaccaattctgttggctgggccgtgatcaccgacgagtacaaggtg

ccgtccaagaagttcaaggtcctcggcaacaccgaccgccactccatcaagaagaatctcatcggcgccctgctgttcgactctggcg

agacagccgaggctacaaggctcaagaggaccgctagacgcaggtacaccaggcgcaagaaccgcatctgctacctccaagagat

cttctccaacgagatggccaaggtggacgacagcttcttccacaggctcgaggagagcttcctcgtcgaggaggacaagaagcacga

gcgccatccgatcttcggcaacatcgtggatgaggtggcctaccacgagaagtacccgaccatctaccacctccgcaagaagctcgtc

gactccaccgataaggccgacctcaggctcatctacctcgccctcgcccacatgatcaagttcaggggccacttcctcatcgagggcg

acctcaacccggacaactccgatgtggacaagctgttcatccagctcgtgcagacctacaaccagctgttcgaggagaacccgatcaa

cgcctctggcgttgacgccaaggctattctctctgccaggctctctaagtcccgcaggctcgagaatctgatcgcccaacttccgggcg

agaagaagaatggcctcttcggcaacctgatcgccctctctcttggcctcaccccgaacttcaagtccaacttcgacctcgccgaggac

gccaagctccagctttccaaggacacctacgacgacgacctcgacaatctcctcgcccagattggcgatcagtacgccgatctgttcct

cgccgccaagaatctctccgacgccatcctcctcagcgacatcctcagggtgaacaccgagatcaccaaggccccactctccgcctc

catgatcaagaggtacgacgagcaccaccaggacctcacactcctcaaggccctcgtgagacagcagctcccagagaagtacaagg

agatcttcttcgaccagtccaagaacggctacgccggctacatcgatggcggcgcttctcaagaggagttctacaagttcatcaagccg

atcctcgagaagatggacggcaccgaggagctgctcgtgaagctcaatagagaggacctcctccgcaagcagcgcaccttcgataat

ggctccatcccgcaccagatccacctcggcgagcttcatgctatcctccgcaggcaagaggacttctacccgttcctcaaggacaacc

gcgagaagattgagaagatcctcaccttccgcatcccgtactacgtgggcccgctcgccaggggcaactccaggttcgcctggatga

ccagaaagtccgaggagacaatcaccccctggaacttcgaggaggtggtggataagggcgcctctgcccagtctttcatcgagcgca

tgaccaacttcgacaagaacctcccgaacgagaaggtgctcccgaagcactcactcctctacgagtacttcaccgtgtacaacgagct

gaccaaggtgaagtacgtgaccgaggggatgaggaagccagctttccttagcggcgagcaaaagaaggccatcgtcgacctgctgtt

caagaccaaccgcaaggtgaccgtgaagcagctcaaggaggactacttcaagaaaatcgagtgcttcgactccgtcgagatctccgg

cgtcgaggataggttcaatgcctccctcgggacctaccacgacctcctcaagattatcaaggacaaggacttcctcgacaacgaggag

aacgaggacatcctcgaggacatcgtgctcaccctcaccctcttcgaggaccgcgagatgatcgaggagcgcctcaagacatacgcc

cacctcttcgacgacaaggtgatgaagcagctgaagcgcaggcgctataccggctggggcaggctctctaggaagctcatcaacgg

catccgcgacaagcagtccggcaagacgatcctcgacttcctcaagtccgacggcttcgccaaccgcaacttcatgcagctcatccac

gacgactccctcaccttcaaggaggacatccaaaaggcccaggtgtccggccaaggcgattccctccatgaacatatcgccaatctcg

ccggctccccggctatcaagaagggcattctccagaccgtgaaggtggtggacgagctggtgaaggtgatgggcaggcacaagcca

gagaacatcgtgatcgagatggcccgcgagaaccagaccacacagaagggccaaaagaactcccgcgagcgcatgaagaggatc

gaggagggcattaaggagctgggctcccagatcctcaaggagcacccagtcgagaacacccagctccagaacgagaagctctacct

ctactacctccagaacggccgcgacatgtacgtggaccaagagctggacatcaaccgcctctccgactacgacgtggaccatattgtg

ccgcagtccttcctgaaggacgactccatcgacaacaaggtgctcacccgctccgacaagaacaggggcaagtccgataacgtgcc

gtccgaagaggtcgtcaagaagatgaagaactactggcgccagctcctcaacgccaagctcatcacccagaggaagttcgacaacct

caccaaggccgagagaggcggcctttccgagcttgataaggccggcttcatcaagcgccagctcgtcgagacacgccagatcacaa

agcacgtggcccagatcctcgactcccgcatgaacaccaagtacgacgagaacgacaagctcatccgcgaggtgaaggtcatcacc

ctcaagtccaagctcgtgtccgacttccgcaaggacttccagttctacaaggtgcgcgagatcaacaactaccaccacgcccacgacg

cctacctcaatgccgtggtgggcacagccctcatcaagaagtacccaaagctcgagtccgagttcgtgtacggcgactacaaggtgta

cgacgtgcgcaagatgatcgccaagtccgagcaagagatcggcaaggcgaccgccaagtacttcttctactccaacatcatgaatttct

tcaagaccgagatcacgctcgccaacggcgagattaggaagaggccgctcatcgagacaaacggcgagacaggcgagatcgtgtg

ggacaagggcagggatttcgccacagtgcgcaaggtgctctccatgccgcaagtgaacatcgtgaagaagaccgaggttcagaccg

gcggcttctccaaggagtccatcctcccaaagcgcaactccgacaagctgatcgcccgcaagaaggactgggacccgaagaagtat

ggcggcttcgattctccgaccgtggcctactctgtgctcgtggttgccaaggtcgagaagggcaagagcaagaagctcaagtccgtca

aggagctgctgggcatcacgatcatggagcgcagcagcttcgagaagaacccaatcgacttcctcgaggccaagggctacaaggag

gtgaagaaggacctcatcatcaagctcccgaagtacagcctcttcgagcttgagaacggccgcaagagaatgctcgcctctgctggcg

agcttcagaagggcaacgagcttgctctcccgtccaagtacgtgaacttcctctacctcgcctcccactacgagaagctcaagggctcc

ccagaggacaacgagcaaaagcagctgttcgtcgagcagcacaagcactacctcgacgagatcatcgagcagatctccgagttctcc

aagcgcgtgatcctcgccgatgccaacctcgataaggtgctcagcgcctacaacaagcaccgcgataagccaattcgcgagcaggc

cgagaacatcatccacctcttcaccctcaccaacctcggcgctccagccgccttcaagtacttcgacaccaccatcgaccgcaagcgct

acacctctaccaaggaggttctcgacgccaccctcatccaccagtctatcacaggcctctacgagacacgcatcgacctctcacaactc

ggcggcgatTCCGGCGGCAGCccaaagaagaagcggaaggtgTCTGGAGGTTCTcctaagaaaaagagaaa

agtgTCCGGCGGCTCCccgaagaagaagcgcaaggtgTGAGAGCTCcggccgggagcatgcgacgtcgatcta

actgactagccgcggccatgctagagtccgcaaaaatcaccagtctctctctacaaatctatctctctctatttttctccagaataatgtgtg

agtagttcccagataagggaattagggttcttatagggtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaa

tacttctatcaataaaatttctaattcctaaaaccaaaatccagtgacct

Drawings

Fig. 1 is a schematic structural diagram of a vector constructed using Nm1Cas9 and SpCas9 according to the present invention;

FIG. 2 is a comparison of the mutant produced by the present invention with a control sequence.

Detailed Description

Embodiments of the present invention are described below with reference to the accompanying drawings. It should be noted that the following examples are only illustrative of exemplary implementations of the present invention and are not intended to limit the present invention in any way. Certain equivalent modifications and obvious improvements to the present invention may be made by those skilled in the art.

The operations in the following detailed description are performed using conventional operations commonly used in the art without additional specificity. The person skilled in the art can easily obtain teachings about such conventional operations from the prior art, for example, see textbooks Sambrook and David Russell, molecular Cloning: A Laboratory Manual,3rd ed., vols1,2; charles Neal Stewart, alicher Touraev, vitaly Citovsky and Tzvi Tzfira, plant Transformation Technologies, etc. The raw materials, reagents, materials and the like used in the following examples are all commercially available products unless otherwise specified.

EXAMPLE 1 construction of expression vectors

The UBI, osSpCas9, osNm1Cas9 and 35S terminator genes were amplified and the T-UBI-OsSpCas9-35S terminator and T-UBI-OsNm1Cas9-35S terminator were assembled by homologous recombination on the intermediate vector T. Connecting UBI-OsSpCas9-35S terminator to a PHUC400 vector existing in a laboratory by using PstI/SacI restriction enzyme site to obtain PHUC-SpCas9, and connecting UBI-OsNm1Cas9-35S terminator to the PHUC-SpCas9 vector by using PmeI restriction enzyme site to obtain PHUC-Sp-Nm1; on this basis, the sgRNA expression cassette was ligated by HindIII cleavage to give the final expression vector PHUC411-Nm1, which is shown in FIG. 1.

Example 2 construction of targeting vectors

A20 bp sequence at the front end of the PAM sequence of NGGNGATT on the rice PDS gene is selected as a target position of SpCas9, such as a fourth exon GCTGCTTGGAAGGATGAAGATGGAGATT sequence, then utilizing SpCas9 tends to produce-44 bit deletion characteristic, nm1Cas9 targeting recognition sequence is GATAGCTGCTTGGAAGGATG-AGATGGAGATTThe underlined part is the PAM sequence. After the identification sequence is determined, a double-targeting primer is synthesized by using an existing intermediate vector MsgScRNA-OsU3 in a laboratory as a template, and amplified and recovered, wherein the primer sequence is as follows:

MScRNA-PDS FP:

AATAATGGTCTCAGGCAGATAGCTGCTTGGAAGGATGAGAGTTGTAGCTCCCTTTCTCATTT

MScRNA-PDS RP:

AATAATGGTCTCAAAACTCTTCATCCTTCCAAGCAGCTGCCACGGATCATCTGCACAACTCTT the above recovered product was fused with the expression vector of PHUC411-Nm1 by GoldGate, plasmids were extracted and sequenced, and the correctly sequenced vector was transferred into Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 strain (kept by the university of Anhui agriculture, agricultural college) for genetic transformation by freeze thawing.

Example 3-acquisition of rice genetic transformation and mutant with double expression cassettes of SpCas9 and NmCas9 as targeting vectors.

1. Induction and preculture of mature embryo callus

Removing shell from mature seeds of Japanese sunny, selecting seeds with normal appearance and clean and no mildew, shaking with 70% alcohol for 90sec, and pouring out alcohol; the seeds were washed with 50% sodium hypochlorite (stock solution available chlorine concentration greater than 4%) containing Tween20, 1 drop of Tween20 was added per 100 ml, and shaken on a shaker for 45min (180 r/min). Pouring out sodium hypochlorite, washing with sterile water for 5-10 times until no sodium hypochlorite smell exists, and finally adding sterile water and soaking overnight at 30 ℃. Embryos were separated along the aleurone layer with a scalpel blade and scutellum placed up on induction medium (composition see table 1), 12 grains/dish, and dark cultured at 30 ℃ to induce callus.

After two weeks, spherical, rough and pale yellow secondary calli appear, and the pre-culture operation can be carried out, namely, the secondary calli are transferred to a new callus induction culture medium, and the secondary calli are subjected to dark culture at 30 ℃ for 5 days. After the preculture is finished, small particles with good states and vigorous division are collected into a 50mL sterile centrifuge tube by a spoon and used for agrobacterium infection.

2. Cultivation of Agrobacterium Strain and suspension preparation

Agrobacterium strain EHA105 containing the targeting vector was streaked on LB plates containing 50mg/L kanamycin (see Table 1 for composition), cultured in the dark at 28℃and after 24h the activated Agrobacterium was inoculated with a sterile inoculating loop onto fresh LB plates of 50mg/L kanamycin for a second activation, and cultured overnight in the dark at 28 ℃. 20-30mL of Agrobacterium suspension medium (composition shown in Table 1) was added to a 50mL sterile centrifuge tube, activated 2 times with an inoculating loop, OD660 was adjusted to about 0.10-0.25, and the mixture was allowed to stand at room temperature for 30min or more.

3. Infection and co-cultivation

To the prepared callus (see step 1), the Agrobacterium suspension was added and soaked for 15min, with gentle shaking in between. Pouring out the liquid after the soaking is finished (the liquid is dripped as much as possible), sucking out the superfluous agrobacterium liquid on the surface of the callus by using sterile filter paper, and drying by using sterile air in an ultra clean bench. Three pieces of sterile filter paper are placed on a disposable sterile petri dish with the thickness of 100 multiplied by 25mm, 2.5mL of agrobacterium suspension medium is added, the callus after the suction is uniformly dispersed on the filter paper, and the callus is cultivated for 48 hours in the dark at the temperature of 23 ℃.

4. Pre-screening and screening culture

After the end of co-cultivation, the co-cultivated calli were evenly spread in pre-screening medium (composition see Table 1), and cultivated in the dark at 30℃for 5 days. After the pre-screening culture is finished, transferring the calli to a screening culture medium (the composition is shown in table 1), inoculating 25 calli to each culture dish, culturing in the dark at 30 ℃, and after 2-3 weeks, obviously growing the resistant calli, and carrying out differentiation and regeneration operation.

5. Differentiation and regeneration

2-3 small fresh particles with good growth state are selected for each independent transformant and transferred to a differentiation regeneration medium (the composition is shown in Table 1). Each dish was inoculated with 5 independent transformants. Culturing at 28deg.C under light with light intensity of 3000-6000lx for 16 hr and 8 hr in dark.

6. Rooting and transplanting

When the buds of the resistant callus grow to about 2cm, only one well-grown seedling is taken from each independent transformant, and the seedlings are transferred to a rooting medium (the composition is shown in table 1), and are cultivated by illumination at 28 ℃ for 16 hours, dark for 8 hours and light intensity of 3000-6000lx. After two weeks, young seedlings with developed root systems are selected, the culture medium is washed off by water, and the young seedlings are transplanted into soil.

Example 4 molecular characterization of Rice genetically transformed plants

Before transplanting, rice leaf samples are taken, and genome DNA of 48 transformed plants is extracted by a CTAB method. The resulting genomic DNA samples were used in HITOM sequencing analysis to detect the frequency of substitution of the site-4 of the target site with A/T in the transformed population. From the HITOM results, it was found that various types of gene mutations occurred in 48 plants for this locus on the fourth exon of rice OsPDS. The plant ratio of the T-4-bit A is 10, the existing single-base editing technology can only realize the C-T or A-G substitution, but can not realize the A-T substitution, and the action site of the guiding editing technology is usually at the-3-bit and the C-terminal, and can not realize the-4-bit substitution. Plants with G replaced by A at the-4 position have a 5-plant ratio, wild-type have a 8-plant ratio, and the rest are single or multiple base deletions. Figure 2 shows a partial mutation situation. At present, no scheme for reporting A-T replacement with high efficiency is known, and the scheme has great significance.

Claims (8)

1. A gene sequence for achieving accurate a/T replacement at the-4 position of a target site, characterized in that the gene sequence comprises a double expression cassette of SpCas9 and NmCas 9.

2. The gene sequence according to claim 1, wherein the double expression cassette is composed of a nucleotide sequence shown as SEQ ID NO.1 of the sequence Listing.

3. An expression cassette comprising the dual SpCas9 and NmCas9 expression cassette of claim 1.

4. An expression vector comprising the gene sequence of claim 1 or the expression cassette of claim 3.

5. Use of the gene of claim 1, the expression cassette of claim 3 or the vector of claim 4, comprising cleaving the rice genome for a PAM sequence NGGNGATT with the double expression cassette to obtain a transgenic plant or plant part comprising an a/T mutation site.

6. A method for inserting a/T mutation sites at a target site of interest using an expression vector comprising the gene sequence of claim 1, comprising:

amplifying UBI, osSpCas9, osNm1Cas9 and 35S terminator genes, and assembling the T-UBI-OsSpCas9-35S terminator and the T-UBI-OsNm1Cas9-35S terminator on an intermediate vector T by a homologous recombination mode;

ligating UBI-OsSpCas9-35S terminator to the gluc 400 vector using the PstI/SacI cleavage site to give gluc-SpCas 9;

ligating the UBI-OsNm1Cas9-35S terminator to the gluc-SpCas 9 vector using a PmeI cleavage site to obtain gluc-Sp-Nm 1; on the basis, a final expression vector PHUC411-Nm1 is obtained by enzyme digestion of HindIII and connection of an sgRNA expression frame;

constructing a targeting vector;

using an agrobacterium strain containing a gluc 411-Nm1 targeting vector for infecting a seed of interest;

co-culturing and screening the obtained callus to obtain edited plant callus, and differentiating the callus into plants.

7. A method for introducing a gene editing tool of the gene sequence of claim 1 into rice cells, comprising transferring a vector containing double expression cassettes of SpCas9 and NmCas9 into agrobacterium tumefaciens, infecting and culturing rice tissues by using agrobacterium tumefaciens, and transferring seedlings obtained by tissue culture into a rooting medium for rooting.

8. The use according to claim 7, characterized in that it comprises the steps of infection, co-cultivation, screening to obtain edited plant calli, then differentiating the calli into plants, detecting the mutation frequency of the substitution of any base at position-4 of the target site with a or T.