阅读说明：本技术 一种植物基因组定向碱基编辑骨架载体及其应用 (Plant genome directed base editing framework vector and application thereof ) 是由 张勇 唐旭 郑雪莲 任秋蓉 周建平 邓科君 于 2018-11-23 设计创作，主要内容包括：本发明属于基因工程技术领域,具体涉及一种植物基因组定向碱基编辑骨架载体及其应用。本发明要解决的技术问题是提升植物细胞基因组的定向碱基编辑效率、拓展碱基编辑窗口。本发明解决技术问题的技术方案是提供一种植物基因组定向碱基编辑骨架载体,该骨架载体由一个PolⅡ型启动子驱动nCas9-PmCDA1核酸酶-胞嘧啶脱氨酶融合蛋白表达单元和合成向导RNA(sgRNA)转录表达单元两个核心区域构成的核心单元的转录。本发明单一转录单元定向碱基编辑骨架载体,可有效实现胞嘧啶碱基(C)转变为胸腺嘧啶碱基(T)的简单、快捷、高效定向编辑,是一种有效实现植物基因组碱基定向编辑的分子工具。(The invention belongs to the technical field of genetic engineering, and particularly relates to a plant genome directional base editing framework vector and application thereof. The invention aims to improve the directional base editing efficiency of plant cell genome and expand the base editing window. The technical scheme for solving the technical problem is to provide a plant genome directed base editing framework vector, wherein the framework vector is used for driving the transcription of a core unit consisting of an nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression unit and a synthetic guide RNA (sgRNA) transcription expression unit by a Pol II type promoter. The single transcription unit directional base editing framework vector can effectively realize simple, quick and efficient directional editing of converting cytosine base (C) into thymine base (T), and is a molecular tool for effectively realizing directional editing of plant genome base.)

1. A plant genome directed base editing backbone vector characterized in that: comprises a core unit consisting of two core regions of an nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression unit and a synthetic guide RNA (sgRNA) transcription expression unit, wherein the core unit is driven by a Pol II type promoter to transcribe;

the core unit is nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-T in sequence from 5 'direction to 3' direction; the nCas9ORF is a coding frame of a Streptococcus pyogenes nuclease protein D10A mutant; PmCDA1 is a functional unit of cytosine deaminase coding region; poly A is Poly A area; the sgRNA cloning and transcription unit is sgRNA cloning and transcription unit, and the sgRNA cloning scaffold is at least one; t is a terminator.

2. The plant genome directed base editing scaffold vector of claim 1, wherein: the functional unit of the coding region of the PmCDA1 cytosine deaminase sequentially comprises a GGGS joint, an SH3 joint, a PmCDA1 coding region, an NLS signal peptide, an UGI coding region, an SGGS joint and an NLS signal peptide from the N end to the C end.

3. The plant genome directed base editing scaffold vector of claim 1 or 2, conforming to at least one of:

a. the amino acid sequence coded by nCas9 nuclease protein D10A mutant coding frame nCas9ORF is shown as amino acids from position 1 to position 1382 in Seq ID No. 2;

b. the amino acid sequence encoded by the functional unit of the cytosine deaminase coding region of PmCDA1 is shown as amino acids 1383 to 1788 in Seq ID No. 2.

4. The plant genome directed base editing scaffold vector according to any one of claims 1 to 3, wherein: the sgRNA cloning and transcription unit sgRNA cloning scaffold comprises a tRNA-Gly coding sequence, a BsaI-ccdB-BsaI unit, an sgRNA framework coding sequence and a tRNA-Gly coding sequence from 5 'end to 3' end in sequence.

5. The plant genome directed base editing scaffold vector of any one of claims 1 to 4, wherein: the number of sgRNA cloning and transcription units is 1-6.

6. The plant genome directed base editing scaffold vector of any one of claims 1 to 5, wherein: the nucleotide sequence of the sgRNA cloning and transcription unit sgRNA cloning scaffold is shown as 7432bp to 8300bp in Seq ID No. 1.

7. The plant genome directed base editing scaffold vector of any one of claims 1 to 7, wherein: at least one of the following is met:

a. the nucleotide sequence encoded by nCas9 nuclease protein D10A mutant encoding frame nCas9ORF is shown as 2011bp to 6156bp in Seq ID No. 1;

b. the nucleotide sequence coded by the functional unit of the coding region of the PmCDA1 cytosine deaminase is shown in 6157bp to 7374bp in Seq ID No. 1;

c. the nucleotide sequence of Poly A region Poly A is shown as 7384bp to 7431bp in Seq ID No. 1.

8. The plant genome directed base editing scaffold vector of any one of claims 1 to 6, wherein:

at least one of the following is met:

a. the terminator is a rice HSP terminator HSPT, and the nucleotide sequence of the terminator is shown by a nucleotide sequence from 8307bp to 8556bp in Seq ID No. 1;

b. the Pol II type promoter is a corn pZmUbi1 promoter pZmUbi1, and the nucleotide sequence is shown from the 1bp to the 2008bp in Seq ID No. 1.

9. The plant genome directed base editing scaffold vector of any one of claims 1 to 8, wherein: the core unit of the framework vector has the structure of pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloningscaffold-HSPT, and the nucleotide sequence is shown in Seq ID No. 1.

10. The preparation method of the recombinant expression vector for carrying out directional base editing aiming at the specific cytosine base of the target site of the plant genome comprises the following steps:

a. defining a target DNA region of a specific biological genome, analyzing the region with PAM characteristics, and selecting a DNA sequence of 15-30 bpadjacent to the 5' end of a PAM structure as a specific target sequence;

b. respectively synthesizing 5' -CGGA-N according to selected specific target sequence_XA forward oligonucleotide strand of-3 'character and having 5' -AAAC-N_X-a reverse oligonucleotide strand of 3' character, N represents any of A, G, C, T, X is an integer, and 14 ≦ X ≦ 30, wherein N in the forward oligonucleotide strand_XAnd N in reverse oligonucleotide_XHas reverse complementary characteristics; obtaining a complementary oligonucleotide double-stranded fragment by annealing;

c. mixing the plant genome directed base editing skeleton vector of any one of claims 1 to 9 with the complementary oligonucleotide double-stranded fragment obtained in step b, simultaneously adding BsaI endonuclease and T4DNA ligase into a reaction system, and setting enzyme digestion-ligation cycling reaction to obtain the recombinant expression vector for site directed base editing.

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a plant genome directed base editing framework vector and application thereof.

Background

Genome directed modification has been the leading and hot field of biological research, and is achieved by precisely directing and modifying specific regions of the genome: on one hand, the method can carry out accurate mutation aiming at a target sequence to obtain a mutant material and definitely identify the function of a target gene; on the other hand, the method can carry out accurate replacement or insertion of a target sequence and minimize the uncertainty of expression and inheritance caused by random introduction of foreign genes.

In 2012, researchers proved for the first time that CRISPR-Cas (Clustered regulated amplified short palindromic repeats-CRISPR associated protein) can realize sequence-specific DNA double-strand splicing, and then CRISPR-Cas9 system realizes RNA-guide-based intracellular genome-directed editing in animal and plant systems including cynomolgus monkey, zebrafish, mouse, human cell line, arabidopsis thaliana, rice and the like. In the genome targeted editing system, under the guidance of guide RNA, Cas protein recognizes and cuts specific DNA sequences to generate DNA Double Strand Breaks (DSBs), and then targeted editing of the DNA sequences of target sites is realized based on a cell endogenous DNA repair system. The eukaryotic DNA repair systems currently known can be divided into two broad categories: repair by "homologous recombination" (HR); "non-homologous end joining" (NHEJ) repair. HR precisely repairs damaged DNA regions by taking homologous sequences as templates; NHEJ does not need the existence of homologous sequence, and the broken ends formed by DNA damage are directly connected, so that sequence variation of different degrees is often introduced while the repair is completed.

Although the CRISPR-Cas genome editing tool can effectively realize targeted editing of a target genome sequence, editing events based on an NHEJ repair pathway mainly introduce base insertion or deletion mutation at a target modification site at random, and although the editing events based on an HR repair pathway can accurately replace the target modification site sequence according to donor template DNA, the occurrence frequency efficiency of the editing events is far lower than that of editing events mediated by an NHEJ repair pathway, so that the effective application of the CRISPR-Cas genome editing tool in precise base editing related basic research and application practice is greatly limited.

In order to improve the accurate editing efficiency of the specific base of the genome target site and effectively realize the accurate replacement editing of the single base of the target site, researchers realize the accurate replacement editing of the specific base of the genome target site (for example, base C is replaced by base T; base A is replaced by base G) by fusing the specific base deaminase with dCas9, nCas9or your Cas12a on the basis of a CRISPR-Cas genome editing tool, and the novel genome editing tool is called a Base Editor (BE). The directional base editing technology can effectively replace and edit a specific single base of a genome target site, is beneficial supplement of a CRISPR-Cas genome editing technology, is judged as one of ten-year-old scientific breakthroughs in 2017 by 'science' journal, and highlights important potential of the technology in basic research and application practice.

The CRISPR-Cas system-based directional base editing tool effectively expands the application range of the CRISPR-Cas system and shows wide application prospects. However, the existing directional base editing tools generally have the problems of low editing efficiency and limited editing window, particularly in plant genome editing application practice, the problems are more obvious, and the development of an enhanced plant directional base editing tool with high base editing efficiency and a wide base editing window is urgently needed, so that the active application of a directional base editing technology based on a CRISPR-Cas system in plant genome function research and breeding practice is effectively expanded.

Disclosure of Invention

The invention aims to improve the directional base editing efficiency of plant cell genome and expand the base editing window.

The technical scheme for solving the technical problem is to provide a plant genome directional base editing skeleton vector. The skeleton vector comprises a core unit consisting of two core regions of an nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression unit and a synthetic guide RNA (sgRNA) transcription expression unit, wherein the core unit is driven by a Pol II type promoter to transcribe;

the core unit is nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-T in sequence from 5 'direction to 3' direction; the nCas9ORF is a coding frame of a Streptococcus pyogenes nuclease protein D10A mutant; PmCDA1 is a functional unit of cytosine deaminase coding region; poly A is Poly A area; the sgRNA cloning and transcription unit is sgRNA cloning and transcription unit, and the sgRNA cloning scaffold is at least one; t is a terminator.

Wherein, the functional unit of the coding region of the PmCDA1 cytosine deaminase in the skeleton vector sequentially comprises a GGGS joint, a SH3 joint, a PmCDA1 coding region, an NLS signal peptide, an UGI coding region, an SGGS joint and an NLS signal peptide from the N end to the C end.

Wherein the above-mentioned skeletal carrier meets at least one of the following:

a. the amino acid sequence coded by nCas9 nuclease protein D10A mutant coding frame nCas9ORF is shown as amino acids from position 1 to position 1382 in Seq ID No. 2;

b. the amino acid sequence encoded by the functional unit of the cytosine deaminase coding region of PmCDA1 is shown as amino acids 1383 to 1788 in Seq ID No. 2.

Wherein, the sgRNA cloning and transcription unit sgRNA cloning scaffold in the framework vector sequentially comprises a tRNA-Gly coding sequence, a BsaI-ccdB-BsaI unit, a sgRNA framework coding sequence and a tRNA-Gly coding sequence from the 5 'end to the 3' end.

Wherein, the number of sgRNA cloning and transcription units in the framework vector is 1-6.

The nucleotide sequence of the sgRNA cloning and transcription unit sgRNA cloning scaffold in the framework vector is shown as 7432bp to 8300bp in Seq ID No. 1.

Wherein the above-mentioned skeletal carrier meets at least one of the following:

a. the nucleotide sequence coded by nCas9 nuclease protein D10A mutant coding frame nCas9ORF is shown as 2011bp to 6156bp in Seq ID No. 1;

b. the nucleotide sequence coded by the functional unit of the coding region of the cytosine deaminase of PmCDA1 is shown as 6157bp to 7374bp in Seq ID No. 1.

c. The nucleotide sequence of Poly A region Poly A is shown from 7384bp to 7431bp in Seq ID No.1

d. The terminator is rice HSP terminator HSP T, and the nucleotide sequence is shown as a nucleotide sequence from 8307bp to 8556bp in Seq ID No. 1.

e. The Pol II type promoter is a corn pZmUbi1 promoter pZmUbi1, and the nucleotide sequence is shown from the 1bp to the 2008bp in Seq ID No. 1.

Wherein the core unit in the framework vector has the structure of pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSPT. Further, the nucleotide sequence of the core unit of pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSP T is shown in Seq ID No. 1.

Based on the skeleton vector, the invention also provides a preparation method of the recombinant expression vector for carrying out directional base editing on the specific cytosine base of the target site of the plant genome. The method comprises the following steps:

a. defining a target DNA region of a specific biological genome, analyzing a region with PAM (PAM full name promoter motif adjacent to a candidate recognition site) characteristic, and selecting a 15-30 bpDNA sequence adjacent to the 5' end of a PAM structure as a specific target sequence;

b. respectively synthesizing 5' -CGGA-N according to selected specific target sequence_XA forward oligonucleotide strand of-3 'character and having 5' -AAAC-N_X-a reverse oligonucleotide strand of 3' character, N represents any of A, G, C, T, X is an integer, and 14 ≦ X ≦ 30, wherein N in the forward oligonucleotide strand_XAnd N in reverse oligonucleotide_XHas reverse complementary characteristics; obtaining a complementary oligonucleotide double-stranded fragment by annealing;

c. mixing the plant genome directed base editing skeleton vector of any one of claims 1 to 9 with the complementary oligonucleotide double-stranded fragment obtained in step b, simultaneously adding BsaI endonuclease and T4DNA ligase into the reaction system, and setting enzyme digestion-ligation cycling reaction to obtain the recombinant expression vector for directed base editing of the site.

Further, the length of the specific target sequence in the step a is 18-21 bp. Preferably, the length of the specific target sequence in step a is 20 bp.

Preferably, in step b, X is 18. ltoreq. X.ltoreq.21.

Preferably, in practical operation, a fusion PCR amplification strategy can be applied in step c to obtain a plurality of sgRNA transcription units that are separated by tRNA sequences and are amplified in series, and a BsaI-ccdB-BsaI unit is replaced by a "BsaI digestion-T4 DNA ligase ligation" cycling reaction, and the multiple sgRNA transcription units are cloned into a sgRNA cloning and transcription unit to obtain a recombinant expression vector that can perform specifically-directed base editing on a plurality of target sites.

The invention has the beneficial effects that: the invention constructs the core unit of a single transcription unit directional base editing framework vector by starting two core regions of a promoter-driven nCas9-PmCDA1 fusion protein and a synthetic guide RNA (sgRNA) transcription expression unit. The directional base editing skeleton vector containing the core unit can effectively realize simple, quick and efficient directional editing of converting cytosine base (C) into thymine base (T) aiming at a plant genome target sequence. Compared with the currently used plant base editing tool, the invention improves the base editing efficiency, expands the base editing window, promotes the effective application of the directional base editing strategy in the directional editing of plant genomes, is a molecular tool for effectively realizing the directional editing of the plant genomes bases, and has good application prospect.

Drawings

FIG. 1 is a schematic diagram of the core unit structure and the working of the single transcription unit directional base editing framework vector of the plant STU nCas9-PmCDA1 of the invention.

FIG. 2 shows targeted site cytosine targeted editing efficiency analysis based on Illumina high-throughput sequencing by transiently transforming rice protoplasts with STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, and STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vectors. Wherein nCas9-PmCDA1 represents the single transcription unit oriented base editing framework vector of the plant STU nCas9-PmCDA1 of the invention, and nCas9-rApobec1 is a control group (according to the reference report (Komor AC, Kim YB, Packer MS, Zuris JA, LiuDR.2016. progrmmable editing of a target base in genomic DNA without double-stranded DNA clean. Nature,533(7603):420-424.), rApobec1 cytosine deaminase is substituted for the PmCDA1 unit in the single transcription unit oriented base editing framework vector of the plant STU nCas9-PmCDA1 of the invention).

FIG. 3 shows that rice protoplasts are transiently transformed based on the STU nCas9-PmCDA1-OsCDC48-sgRNA01 recombinant expression vector of the invention, Illumina high-throughput sequencing is performed, and the editing efficiency analysis of replacement of cytosine base sites at different positions of a specific editing site into thymine bases is performed. Wherein nCas9-PmCDA1 and nCas9-rApobec1 are as illustrated in FIG. 2.

FIG. 4 shows that rice protoplasts are transiently transformed based on the STU nCas9-PmCDA1-OsROC5-gRNA04 recombinant expression vector of the invention, Illumina high-throughput sequencing is performed, and the editing efficiency analysis of replacement of cytosine base sites at different positions of specific editing sites into thymine bases is performed. Wherein nCas9-PmCDA1 and nCas9-rApobec1 are as illustrated in FIG. 2.

FIG. 5 shows that rice protoplasts are transiently transformed based on the STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vector of the invention, Illumina high-throughput sequencing is performed, and the editing efficiency analysis of replacement of cytosine base sites at different positions of specific editing sites into thymine bases is performed. Wherein nCas9-PmCDA1 and nCas9-rApobec1 are as illustrated in FIG. 2.

FIG. 6 shows the results of Agrobacterium-mediated rice genetic transformation based on the recombinant expression vectors of STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04 and STU nCas9-PmCDA1-OsROC5-gRNA05, extraction of rice transformation regeneration seedling genome DNA, PCR amplification and Sanger sequencing analysis, and targeted site cytosine targeted editing efficiency analysis.

Detailed Description

The invention constructs a plant genome directed base editing framework vector (also called plant STU nCas9-PmCDA1 single transcription unit directed base editing framework vector in the invention) based on a CRISPR-Cas9 single transcription system and PmCDA1 cytosine deaminase through strategies such as coding region codon optimization, functional unit multiplex assembly and the like,

the plant genome directed base editing framework vector comprises a core unit consisting of two core regions of an nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression unit and a synthetic guide RNA (sgRNA) transcription expression unit, wherein the core unit is driven to be transcribed by a Pol II type promoter;

the nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression unit comprises an nCas9ORF as a coding frame of a Streptococcus pyogenes nuclease protein D10A mutant; PmCDA1 is a functional unit of cytosine deaminase coding region; poly A is Poly A region, namely nCas9 ORF-PmCDA1-Poly A;

the core unit is nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-T in sequence from 5 'direction to 3' direction; the nCas9ORF is a coding frame of a Streptococcus pyogenes nuclease protein D10A mutant; PmCDA1 is a functional unit of cytosine deaminase coding region; poly A is Poly A area; the sgRNA cloning and transcription unit is sgRNA cloning and transcription unit, and the sgRNA cloning scaffold is at least one; t is a terminator.

Wherein, the functional unit of the coding region of the PmCDA1 cytosine deaminase in the skeleton vector sequentially comprises a GGGS joint, a SH3 joint, a PmCDA1 coding region, an NLS signal peptide, an UGI coding region, an SGGS joint and an NLS signal peptide from the N end to the C end.

Wherein the above-mentioned skeletal carrier meets at least one of the following:

a. the amino acid sequence coded by nCas9 nuclease protein D10A mutant coding frame nCas9ORF is shown as amino acids from position 1 to position 1382 in Seq ID No. 2;

b. the amino acid sequence encoded by the functional unit of the cytosine deaminase coding region of PmCDA1 is shown as amino acids 1383 to 1788 in Seq ID No. 2. The two components are connected to form an nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression frame, and the amino acid sequence is shown as Seq ID No.2 in the sequence table.

Wherein, the sgRNA cloning and transcription unit sgRNA cloning scaffold in the framework vector sequentially comprises a tRNA-Gly coding sequence, a BsaI-ccdB-BsaI unit, a sgRNA framework coding sequence and a tRNA-Gly coding sequence from the 5 'end to the 3' end.

Wherein, the number of sgRNA cloning and transcription units in the framework vector is 1-6.

The nucleotide sequence of the sgRNA cloning and transcription unit sgRNA cloning scaffold in the framework vector is shown as 7432bp to 8300bp in Seq ID No. 1.

Wherein the above-mentioned skeletal carrier meets at least one of the following:

a. the nucleotide sequence coded by nCas9 nuclease protein D10A mutant coding frame nCas9ORF is shown as 2011bp to 6156bp in Seq ID No. 1;

b. the nucleotide sequence coded by the functional unit of the coding region of the cytosine deaminase of PmCDA1 is shown as 6157bp to 7374bp in Seq ID No. 1.

c. The nucleotide sequence of Poly A region Poly A is shown from 7384bp to 7431bp in Seq ID No.1

d. The terminator is rice HSP terminator HSP T, and the nucleotide sequence is shown as a nucleotide sequence from 8307bp to 8556bp in Seq ID No. 1.

e. The Pol II type promoter is a corn pZmUbi1 promoter pZmUbi1, and the nucleotide sequence is shown from the 1bp to the 2008bp in Seq ID No. 1.

Wherein the core unit in the framework vector has the structure of pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSPT. Further, the nucleotide sequence of the core unit of pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSP T is shown in Seq ID No. 1.

The core unit (pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSPT) can replace a promoter and a terminator in the promoter unit according to the requirements of a specific transformed host organism and experiments with any Pol II type promoter element (such as promoter elements commonly used in plants, such as OsUb1, CaMV35S and AtUb 10) and terminator element (such as terminator elements commonly used in plants, such as Nos T and 35s T) and can be placed in any plant expression skeleton vector (such as vector series commonly used in plants, such as pCambia, pBI, pMDC, pGreen and the like) to realize site-specific directed base editing.

In the invention, based on a single transcription unit directional base editing framework vector of a plant STU nCas9-PmCDA1, a specific plant genome site-specific STU nCas9-PmCDA1-sgRNA directional base editing recombinant expression vector is constructed and transformed, and under the condition of living cells, a Pol II promoter drives 'nCas 9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold' to be transcribed as a whole transcription unit to obtain a single-chain primary transcript. Under the action of a cell endogenous tRNA processing factor, single primary transcripts are subjected to self-shearing at two tRNA sites respectively to obtain complete nCas9 ORF-PmCDA1 nuclease-cytosine deaminase fusion protein expression frame mRNA (containing Poly A) and sgRNA transcription units. In a cell system, an nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression frame (containing Poly A) is further translated to obtain nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein, and the nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein is combined with an existing sgRNA unit to form a functional nCas9-PmCDA1-sgRNA composite unit for genome target site specific cytosine base directed editing.

In the invention, the complete sgRNA is formed by replacing a BsaI-ccdB-BsaI unit in a sgRNA cloning and transcription unit of a framework vector by an 18-21 bp RNA fragment which can be complementarily combined with the target fragment, the framework RNA fragment is formed by embedding sgRNA, tracrRNA and crRNA which can be combined with a protospacer site in sequence to form functional RNA similar to a hairpin structure, and the framework RNA fragment can be combined with Cas9 nuclease.

After the sgRNA site is determined for a specific target gene (5' -N)_X-NGG-3'; n represents A, G, C, T, X is an integer, and 14 is more than or equal to X is less than or equal to 30(18, 19, 20 and 21 are common values)), according to the construction method of the STU nCas9-PmCDA1-sgRNA recombinant expression vector provided by the invention, a designed sgRNA specific target sequence (protospacer) is cloned into a gRNA cloning and transcription unit in a mode of connection 'circulation reaction' of BsaI enzyme digestion-T4 DNA ligase, so that a specific functional STU nCas9-PmCDA1-sgRNA recombinant expression vector is obtainedAnd (3) a body.

In the invention, a BsaI-ccdB-BsaI unit is fused at the sgRNA cloning transcription framework unit end, and the BsaI-ccdB-BsaI unit is used as a multi-cloning site for enzyme digestion of CRISPR/Cas9 single transcription unit framework vector so as to clone a target gRNA specific target sequence (protospacer). The key content of the invention can be effectively realized by replacing a BsaI-ccdB-BsaI unit with a restriction enzyme which can introduce a cut on the framework vector of the invention and correspondingly modifying the cloning site of the sgRNA specific target sequence.

In the process of constructing the plant genome site specificity STU nCas9-PmCDA1-sgRNA directional base editing recombinant expression vector, recombinant clones containing the correct Cas9-gRNA expression vector can be screened through transforming escherichia coli and bacterial screening pressure, and can be identified by colony PCR, plasmid enzyme digestion, sequence determination and other modes, so that the plant genome site specificity STU nCas9-PmCDA1-sgRNA directional base editing recombinant expression vector for the purpose is definitely obtained.

By applying a fusion PCR amplification strategy, a plurality of sgRNA transcription units which are separated by tRNA sequences can be obtained to be connected in series to amplify products, BsaI enzyme digestion-T4 DNA ligase connection is carried out in a circulating reaction mode to replace BsaI-ccdB-BsaI units, the multiple sgRNA transcription units can be cloned into sgRNA cloning and transcription units, and an STU nCas9-PmCDA1-sgRNA1-sgRNA2- … -sgRNA x recombinant expression vector which can be specifically modified aiming at a plurality of target sites is obtained (see figure 1). Preferably, the number of sgRNA cloning and transcription units in the backbone vector is 1 to 6.

In the invention, site-specific STU nCas9-PmCDA1-sgRNA directed base editing recombinant expression vector constructed according to the invention can be transferred into plant cells by protoplast, gene gun and agrobacterium-mediated multiple transformation methods, so that the transformed cells simultaneously have nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein and sgRNA units aiming at specific genome target sequences; under the combined action of nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein and sgRNA unit, specific cytosine base of a specific genome target sequence is edited (the cytosine base is replaced by T (high probability), A (low probability) and G (low probability)). When the single transcription unit directional base editing framework vector of the STU nCas9-PmCDA1 is applied to plants, resistance genes comprising kanamycin, hygromycin, basta and the like can be used for screening plant transformants, and cells or tissues (such as protoplasts or callus tissues) of positive transformants are differentiated and regenerated to obtain regenerated plants containing target site directional modification.