CN110607320A - Plant genome directed base editing framework vector and application thereof - Google Patents

Abstract

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.

Description

Plant genome directed base editing framework vector and application thereof

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.

Example 1 construction of a plant STU nCas9-PmCDA1 Single transcription Unit directed base editing backbone vector

The invention designs an STU nCas9-PmCDA1 single transcription unit directional base editing framework vector for plant genome engineering, and the core unit of the vector is pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSPT in sequence from 5 'to 3'. Wherein: pZUbi 1, maize pZUbi 1 promoter (the substitution of different PolII type promoters can be realized by the scheme of single transcription unit directional base editing framework vector of AscI, SbfI double digestion basic STU nCas9-PmCDA 1); the nCas9ORF is the coding frame of Streptococcus pyogenes (Streptococcus pyogenes) nuclease protein D10A mutant (containing a C-terminal NLS signal peptide); the PmCDA1 is a functional unit of a cytosine deaminase coding region (sequentially comprising 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); poly A is Poly A area; a sgRNA cloning and transcription unit (comprising a tRNA-Gly coding sequence, a BsaI-ccdB-BsaI unit, a sgRNA framework coding sequence and a tRNA-Gly coding sequence from a 5 'end to a 3' end); HSP T is rice HSP terminator (the replacement of different terminators can be realized by a scheme of directional base editing of a framework vector by a single transcription unit of BamHI and SacI double-enzyme digestion basic STUnCas9-PmCDA 1). The structure and the working principle of the single transcription unit directional base editing framework vector of the plant STU nCas9-PmCDA1 are shown in figure 1.

Optionally, the scaffold carrier further comprises: the left and right border sequences of the T-DNA, "pZmUbi 1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSPT" core unit is located between the left and right borders of the T-DNA; the T-DNA can also comprise a hygromycin resistance gene expression unit (sequentially comprising the elements of 2 xCaMV 35S promoter-hygromycin phosphotransferase ORF-CaMV poly A; and the replacement of different resistance gene ORFs can be realized through the scheme of an AvrII and PacI double-enzyme digestion basic CRISPR/Cas9 single transcription unit framework vector) between the left and right border sequences as a plant transformant screening marker.

In order to realize the quick and efficient construction of a specific genome target STU nCas9-PmCDA1-sgRNA directed base editing recombinant expression vector, the plant STU nCas9-PmCDA1 single transcription unit directed base editing skeleton vector is fused with 637bp BsaI-ccdB-BsaI unit at the 5' end of the sgRNA transcription expression unit, based on the design strategy, in the subsequent construction process of the target STU nCas9-PmCDA1-sgRNA directional base editing recombinant expression vector, only needs to mix the plant STU nCas9-PmCDA1 single transcription unit directional base editing framework vector, the annealed specific target sequence complementary oligonucleotide double-stranded fragment, BsaI endonuclease and T4DNA ligase in a construction system, and setting a circulating reaction of' enzyme digestion at 37 ℃ and connection at 16 ℃ to realize effective construction of a specific Cas9-gRNA expression vector. The vector can be constructed by the conventional method in the existing molecular cloning technology, and it should be noted that the above element sequence is a unique part of the backbone plasmid vector, and it can also include the general structure of some conventional vectors, which will not be described in the present invention.

Plant codon optimization (adding NLS signal at 3' end) is carried out based on Streptococcus pyogenes (Streptococcus pyogenes) Cas9 nuclease protein coding genes (Streptococcus pyogenes Cas9, SpCas9), D10A mutation is introduced, and the complete ORF sequence of nCas9 nuclease protein D10A mutant coding frame (containing C-end NLS coding sequence) is artificially synthesized, wherein the nucleotide sequence is shown as 2011bp to 6156bp in Seq ID No.1 (coded by the NLS coding sequence)Amino acid sequences such as1AA to 1382AA in Seq ID No. 2). Meanwhile, the sequence (Nishida K, Arazoe T, Yache N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A.2016. targettednucleic acid injection using hybrid prokaryotic and verterbric adaptive immune systems science,353 6305. pii: aaf8729) was examined based on the Lampetra eel (Petrymzon marinus) cytosine deaminase (PmCDA1), and a PmCDA1 cytosine deaminase expression unit coding frame (from N-terminal to C-terminal) was designedThe gene sequence 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), plant codon optimization is carried out, artificial synthesis is carried out, and the nucleotide sequence is shown as 6157bp to 7374bp in Seq ID No.1 (the coded amino acid sequence is shown as 1383AA to 1788AA in Seq ID No. 2). Further, another 3 basic units are obtained by artificial synthesis:

a. frag-A: poly A, nucleotide sequence as shown in 7384bp to 7431bp of Seq ID No. 1;

b. frag-B: the sgRNA cloning and transcription unit coding sequence sequentially comprises a tRNA-Gly coding sequence, a BsaI-ccdB-BsaI unit, a sgRNA framework coding sequence and a tRNA-Gly coding sequence from a 5 'end to a 3' end: the nucleotide sequence is shown as 7432bp to 8300bp in Seq ID No. 1);

c. frag-C: the rice HSP terminator coding sequence has the nucleotide sequence shown as 8307bp to 8556bp in Seq ID No. 1.

Sequentially fusing the coding sequence of the nCas9 nuclease protein D10A mutant coding frame, the coding sequence of the PmCDA1 cytosine deaminase expression unit coding frame and frag-A, frag-B, frag-C in a fusion PCR mode, and respectively adding SbfI and SacI restriction enzyme cutting sites at the 5 'and 3' ends of a fusion PCR product to obtain a 6560bp assembly unit.

SbfI and SacI double digestion are respectively carried out on a vector framework pGEL026(Tang X, Zheng X, Qi YP, Zhang D, Cheng Y, Tang A, Voytas DF, Zhang Y.2016.A single transcript CRISPR-Cas9 system for expression in molecular Plant,9(7): 1088. sup. and 1091.) and a 6560bp assembly unit, and target fragments are recovered, connected and transformed. Colony PCR, plasmid restriction enzyme digestion and DNA sequencing are carried out on the screened positive clones to confirm that 6560bp assembly units are cloned into the downstream of the original pZmUbi1 of pGEL026, and the construction of the plant STU nCas9-PmCDA1 single transcription unit directional base editing framework vector is completed.

Example 2 high-throughput identification of the editing efficiency of cytosine-oriented bases of rice endogenous genes based on the STU nCas9-PmCDA1 system

1. Rice endogenous gene sgRNA design and construction of STU nCas9-PmCDA1 base editing recombinant expression vector

5 '-NGG-3' PAM locus was searched for coding genes of rice OsCDC48(LOC _ Os03g05730) and OsROC5(LOC _ Os02g45250), and a 20bp upstream sequence of PAM was selected to design sgRNA (Table 1).

TABLE 1 design, Synthesis and detection information of endogenous Gene sgRNAcrRNA in Rice

According to the designed nucleic acid sequence of the sgRNA locus, corresponding forward and reverse oligonucleotide chains are artificially synthesized, and the specific sequences are as follows (the upper case base sequence represents the designed locus specificity guide sgRNA locus; the lower case base sequence represents the complementary cohesive end of the framework vector):

BE-OsCDC48-sgRNA01-F(Seq ID No.10)：tgcaGACCAGCCAGCGTCTGGCGC；

BE-OsCDC48-sgRNA01-R(Seq ID No.11)：aaacGCGCCAGACGCTGGCTGGTC；

BE-OsROC5-gRNA04-F(Seq ID No.12)：tgcaGCAGCTGGCTGAGGGTGCAT；

BE-OsROC5-gRNA04-R(Seq ID No.13)：aaacATGCACCCTCAGCCAGCTGC；

BE-OsROC5-gRNA05-F(Seq ID No.14)：tgcaAGCCAGCTGCTTACAAAAC；

BE-OsROC5-gRNA05-R(Seq ID No.15)：aaacGTTTTGTAAGCAGCTGGCT。

BE-OsCDC48-sgRNA01-F/R, BE-OsROC5-gRNA04-F/R, BE-OsROC5-gRNA05-F/R are mixed in equal proportion, boiled water bath is carried out for 10min, and then natural cooling and annealing are carried out to form double-stranded DNA with sticky ends, which is used as an insert fragment for constructing a recombinant vector. Adding plant STU nCas9-PmCDA1 single transcription unit directional base editing skeleton vector, sticky end insert, BsaI endonuclease and T4DNA ligase into a 200uL PCR tube, performing enzyme digestion at 37 ℃ and connection at 16 ℃ for 10 circulation reactions, treating inactivated endonuclease and ligase at 80 ℃, and taking a reaction product to perform escherichia coli transformation.

Positive transformants were identified by kanamycin resistance selection, colony PCR and enzyme digestion, and finally, recombinant expression vectors of STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, and STUnCas9-PmCDA1-OsROC5-gRNA05 were obtained by sequencing and verification, respectively.

2. Rice protoplast transformation of rice endogenous gene STU nca 9-PmCDA1-sgRNA base editing recombinant expression vector

Rice Nipponbare protoplasts are separated, and rice protoplast transformation of recombinant expression vectors of STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04 and STU nCas9-PmCDA1-OsROC5-gRNA05 is respectively carried out based on a PEG-mediated transformation method. Specific procedures for rice protoplast transformation can be found in the experimental procedures disclosed in the literature (Tang X, Zheng X, Qi YP, Zhang D, Cheng Y, Tang A, Voytas DF, Zhang Y.2016. origin transfer CRISPR-Cas9 system for expression genome expression, molecular Plant,9(7): 1088. sup. 1091.).

3. Directional editing and detecting of specific site cytosine bases of endogenous OsCDC48 and OsROC5 genes of rice

After transformation of rice protoplasts, culturing at 25 ℃ in the dark for 48 hours, collecting transformed cells, extracting genome DNA of the rice protoplasts by a CTAB method, and performing PCR amplification and Illumina high-throughput sequencing by using the DNA as a template, wherein the method is disclosed in the specific method reference documents (Tang X, Lowder LG, Zhang T, Malzahn A, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, LiQ, Kirkland ER, Zhang Y, Qi Y.2017.A CRISPR-Cpf1 system for effective genetic evaluation and transcriptional expression in Plants Nature Plants,3: 17018.).

Analysis of Illumina high-throughput sequencing results shows that the editing efficiency of 28.62% (OsCDC48-sgRNA01), 30.99% (OsROC5-gRNA04) and 49.41% (OsROC5-gRNA05) of cytosine base to thymine base is realized for editing target sequences by using 3 cytosine bases of the rice OsCDC48 and OsROC5 endogenous genes respectively (FIG. 2: nCas9-PmCDA 1). Specifically, as a control group (according to a reference report (Komor AC, Kim YB, PackerMS, Zurics JA, Liu DR.2016.programmable editing of a target base in genomic DNAwith double-stranded DNA deletion. Nature,533(7603):420-424.), the rApobec1 cytosine deaminase was substituted for the PmCDA1 unit in the single transcription unit directional base editing framework vector of the plant STU nCas9-PmCDA1 constructed in the present invention), the cytosine editing efficiencies of the same target sequence were 7.65% (OsCDC48-sgRNA01), 4.44% (OsROC5-gRNA04), and 4.16% (OsROC5-gRNA05), respectively (FIG. 2: nCas9-rApobec 1).

Aiming at the determined rice 3 cytosine base editing target sequences, according to Illumina high-throughput sequencing results, further analyzing the editing efficiency of replacing independent cytosine base sites at specific editing sites with thymine bases shows that: the OsCDC48-sgRNA01 has effective thymine base substitution editing of 6 cytosine bases including C3, C4, C7, C8, C11 and C14 (FIG. 3: nCas9-PmCDA 1); the OsROC5-gRNA04 has effective thymine base substitution editing of 2 cytosine bases in total of C2 and C5 (figure 4: nCas9-PmCDA 1); the OsROC5-gRNA05 has effective thymine base substitution editing on 3 cytosine bases of C-1, C3 and C4 (figure 5: nCas9-PmCDA 1). Particularly, based on the single transcription unit directional base editing framework vector of the plant STU nCas9-PmCDA1 constructed by the invention, the editing efficiency of replacing 37.94 percent of cytosine bases with thymine bases at a C-1 site of-1 bp at the position of OsROC5-gRNA05 is detected (figure 5: nCas9-PmCDA1), and the cytosine base editing event positioned outside the sgRNA targeting sequence interval cannot be realized in the existing research. Meanwhile, as a control group, the editing window and editing efficiency of replacing independent cytosine base sites with thymine bases at the tested 3 rice cytosine base editing target sequences are both obviously lower than those of the embodiment of the invention (FIG. 3: nCas9-rApobec 1; FIG. 4: nCas9-rApobec 1; FIG. 5: nCas9-rApobec 1).

The test results show that the single transcription unit directional base editing skeleton vector of the plant STU nCas9-PmCDA1 constructed based on the invention can effectively realize efficient directional editing of the cytosine base in the specific region of the endogenous gene of rice and can further expand the editing window range.

Example 3 creation and efficiency analysis of Rice regenerated plants with cytosine-directed base editing of endogenous Gene of Rice based on STU nCas9-PmCDA1 System

1. Agrobacterium transformation of rice endogenous gene STU nca 9-PmCDA1-sgRNA base editing recombinant expression vector

The recombinant expression vectors successfully constructed in example 2 and tested for directional modification activity in rice protoplasts, STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, and STU nCas9-PmCDA1-OsROC5-gRNA05 were transformed into Agrobacterium EHA105 competent cells by heat shock method, spread on LB solid medium containing 50 mg/L kanamycin and 50 mg/L rifampicin, and cultured in the dark at 28 ℃ for 2 days to obtain positive clones. Positive clones were activated in LB liquid medium containing 50 mg/L kanamycin and 50 mg/L rifampicin for subsequent transformation.

2. Agrobacterium-mediated rice callus transformation of rice endogenous gene STU nCas9-PmCDA1-sgRNA base editing recombinant expression vector

The rice callus transformation is respectively carried out on the recombinant expression vectors of STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04 and STU nCas9-PmCDA1-OsROC5-gRNA05 by an agrobacterium-mediated transformation method. Specific procedures for transformation reference (Tang X, Zheng X, Qi YP, Zhang D, Cheng Y, TangA, Voytas DF, Zhang Y.2016.A single transcript CRISPR-Cas9 system for expression. molecular Plant,9(7): 1088-.

3. Directional base editing detection and efficiency analysis of rice endogenous gene STU nCas9-PmCDA1-sgRNA base editing recombinant expression vector stable transformation regeneration plant

And after transformation, inducing the resistance callus into rice seedlings, extracting the genome DNA of the rice transformation regeneration seedlings, and performing PCR amplification and Sanger sequencing analysis by taking the DNA as a template. Analysis of STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04 and STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vector rice transformation regeneration plants shows that the base editing efficiencies of 44.44% (OsCDC48-sgRNA 01: 8/18), 100.00% (OsROC5-gRNA 04: 26/26) and 68.75% (OsROC5-gRNA 05: 11/16) are respectively realized for 3 cytosine base editing target sequences of the endogenous genes of rice OsCDC48 and OsROC5 (FIG. 6: nCas9-PmCDA 1). The test result further indicates that the single transcription unit directional base editing skeleton vector of the plant STU nCas9-PmCDA1 constructed by the invention can effectively realize efficient directional editing of cytosine bases in a specific region of an endogenous gene of rice and obtain a base editing regeneration plant, is a molecular tool for effectively realizing directional editing of plant genome bases, and can improve the improvement efficiency of genome engineering crops.

Nucleotide and amino acid sequences

Seq ID No. 1: pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSPT nucleotide sequence

CGCGCCTGCAGTGCAGCGTGACCCGGTCGTGCCCCTCTCTTGAGATAATGAGCATTGCATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTGCAGTTTATCTATCTTTATACATATATTTAAACTTTACTCTACGAATAATATAATCTATAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTAGTGTGCATGTGTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCATCCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTTATAGACTAATTTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAAATTAAGAAAACTAAAACTCTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACGGCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGGCGGAGCGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGACACCCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACACACAACCAGAACTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACGCCGCTCGTCCTCCCCCCCCCCCCCTCTCTACCTTCTCAAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTGTGTTAGATCCGTGCTACTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGGAATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTGTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCGGGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCAAGATCGGAGTAGAATTAATTCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTGATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTTCAAGATCGGAGTAGAATACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATATACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCTGCAGCCTGCAGGATGGATAAGAAGTACTCTATCGGACTCGCTATCGGAACTAACTCTGTGGGATGGGCTGTGATCACCGATGAGTACAAGGTGCCATCTAAGAAGTTCAAGGTTCTCGGAAACACCGATAGGCACTCTATCAAGAAAAACCTTATCGGTGCTCTCCTCTTCGATTCTGGTGAAACTGCTGAGGCTACCAGACTCAAGAGAACCGCTAGAAGAAGGTACACCAGAAGAAAGAACAGGATCTGCTACCTCCAAGAGATCTTCTCTAACGAGATGGCTAAAGTGGATGATTCATTCTTCCACAGGCTCGAAGAGTCATTCCTCGTGGAAGAAGATAAGAAGCACGAGAGGCACCCTATCTTCGGAAACATCGTTGATGAGGTGGCATACCACGAGAAGTACCCTACTATCTACCACCTCAGAAAGAAGCTCGTTGATTCTACTGATAAGGCTGATCTCAGGCTCATCTACCTCGCTCTCGCTCACATGATCAAGTTCAGAGGACACTTCCTCATCGAGGGTGATCTCAACCCTGATAACTCTGATGTGGATAAGTTGTTCATCCAGCTCGTGCAGACCTACAACCAGCTTTTCGAAGAGAACCCTATCAACGCTTCAGGTGTGGATGCTAAGGCTATCCTCTCTGCTAGGCTCTCTAAGTCAAGAAGGCTTGAGAACCTCATTGCTCAGCTCCCTGGTGAGAAGAAGAACGGACTTTTCGGAAACTTGATCGCTCTCTCTCTCGGACTCACCCCTAACTTCAAGTCTAACTTCGATCTCGCTGAGGATGCAAAGCTCCAGCTCTCAAAGGATACCTACGATGATGATCTCGATAACCTCCTCGCTCAGATCGGAGATCAGTACGCTGATTTGTTCCTCGCTGCTAAGAACCTCTCTGATGCTATCCTCCTCAGTGATATCCTCAGAGTGAACACCGAGATCACCAAGGCTCCACTCTCAGCTTCTATGATCAAGAGATACGATGAGCACCACCAGGATCTCACACTTCTCAAGGCTCTTGTTAGACAGCAGCTCCCAGAGAAGTACAAAGAGATTTTCTTCGATCAGTCTAAGAACGGATACGCTGGTTACATCGATGGTGGTGCATCTCAAGAAGAGTTCTACAAGTTCATCAAGCCTATCCTCGAGAAGATGGATGGAACCGAGGAACTCCTCGTGAAGCTCAATAGAGAGGATCTTCTCAGAAAGCAGAGGACCTTCGATAACGGATCTATCCCTCATCAGATCCACCTCGGAGAGTTGCACGCTATCCTTAGAAGGCAAGAGGATTTCTACCCATTCCTCAAGGATAACAGGGAAAAGATTGAGAAGATTCTCACCTTCAGAATCCCTTACTACGTGGGACCTCTCGCTAGAGGAAACTCAAGATTCGCTTGGATGACCAGAAAGTCTGAGGAAACCATCACCCCTTGGAACTTCGAAGAGGTGGTGGATAAGGGTGCTAGTGCTCAGTCTTTCATCGAGAGGATGACCAACTTCGATAAGAACCTTCCAAACGAGAAGGTGCTCCCTAAGCACTCTTTGCTCTACGAGTACTTCACCGTGTACAACGAGTTGACCAAGGTTAAGTACGTGACCGAGGGAATGAGGAAGCCTGCTTTTTTGTCAGGTGAGCAAAAGAAGGCTATCGTTGATCTCTTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTCAAAGAGGATTACTTCAAGAAAATCGAGTGCTTCGATTCAGTTGAGATTTCTGGTGTTGAGGATAGGTTCAACGCATCTCTCGGAACCTACCACGATCTCCTCAAGATCATTAAGGATAAGGATTTCTTGGATAACGAGGAAAACGAGGATATCTTGGAGGATATCGTTCTTACCCTCACCCTCTTTGAAGATAGAGAGATGATTGAAGAAAGGCTCAAGACCTACGCTCATCTCTTCGATGATAAGGTGATGAAGCAGTTGAAGAGAAGAAGATACACTGGTTGGGGAAGGCTCTCAAGAAAGCTCATTAACGGAATCAGGGATAAGCAGTCTGGAAAGACAATCCTTGATTTCCTCAAGTCTGATGGATTCGCTAACAGAAACTTCATGCAGCTCATCCACGATGATTCTCTCACCTTTAAAGAGGATATCCAGAAGGCTCAGGTTTCAGGACAGGGTGATAGTCTCCATGAGCATATCGCTAACCTCGCTGGATCTCCTGCAATCAAGAAGGGAATCCTCCAGACTGTGAAGGTTGTGGATGAGTTGGTGAAGGTGATGGGAAGGCATAAGCCTGAGAACATCGTGATCGAAATGGCTAGAGAGAACCAGACCACTCAGAAGGGACAGAAGAACTCTAGGGAAAGGATGAAGAGGATCGAGGAAGGTATCAAAGAGCTTGGATCTCAGATCCTCAAAGAGCACCCTGTTGAGAACACTCAGCTCCAGAATGAGAAGCTCTACCTCTACTACCTCCAGAACGGAAGGGATATGTATGTGGATCAAGAGTTGGATATCAACAGGCTCTCTGATTACGATGTTGATCATATCGTGCCACAGTCATTCTTGAAGGATGATTCTATCGATAACAAGGTGCTCACCAGGTCTGATAAGAACAGGGGTAAGAGTGATAACGTGCCAAGTGAAGAGGTTGTGAAGAAAATGAAGAACTATTGGAGGCAGCTCCTCAACGCTAAGCTCATCACTCAGAGAAAGTTCGATAACTTGACTAAGGCTGAGAGGGGAGGACTCTCTGAATTGGATAAGGCAGGATTCATCAAGAGGCAGCTTGTGGAAACCAGGCAGATCACTAAGCACGTTGCACAGATCCTCGATTCTAGGATGAACACCAAGTACGATGAGAACGATAAGTTGATCAGGGAAGTGAAGGTTATCACCCTCAAGTCAAAGCTCGTGTCTGATTTCAGAAAGGATTTCCAATTCTACAAGGTGAGGGAAATCAACAACTACCACCACGCTCACGATGCTTACCTTAACGCTGTTGTTGGAACCGCTCTCATCAAGAAGTATCCTAAGCTCGAGTCAGAGTTCGTGTACGGTGATTACAAGGTGTACGATGTGAGGAAGATGATCGCTAAGTCTGAGCAAGAGATCGGAAAGGCTACCGCTAAGTATTTCTTCTACTCTAACATCATGAATTTCTTCAAGACCGAGATTACCCTCGCTAACGGTGAGATCAGAAAGAGGCCACTCATCGAGACAAACGGTGAAACAGGTGAGATCGTGTGGGATAAGGGAAGGGATTTCGCTACCGTTAGAAAGGTGCTCTCTATGCCACAGGTGAACATCGTTAAGAAAACCGAGGTGCAGACCGGTGGATTCTCTAAAGAGTCTATCCTCCCTAAGAGGAACTCTGATAAGCTCATTGCTAGGAAGAAGGATTGGGACCCTAAGAAATACGGTGGTTTCGATTCTCCTACCGTGGCTTACTCTGTTCTCGTTGTGGCTAAGGTTGAGAAGGGAAAGAGTAAGAAGCTCAAGTCTGTTAAGGAACTTCTCGGAATCACTATCATGGAAAGGTCATCTTTCGAGAAGAACCCAATCGATTTCCTCGAGGCTAAGGGATACAAAGAGGTTAAGAAGGATCTCATCATCAAGCTCCCAAAGTACTCACTCTTCGAACTCGAGAACGGTAGAAAGAGGATGCTCGCTTCTGCTGGTGAGCTTCAAAAGGGAAACGAGCTTGCTCTCCCATCTAAGTACGTTAACTTTCTTTACCTCGCTTCTCACTACGAGAAGTTGAAGGGATCTCCAGAAGATAACGAGCAGAAGCAACTTTTCGTTGAGCAGCACAAGCACTACTTGGATGAGATCATCGAGCAGATCTCTGAGTTCTCTAAAAGGGTGATCCTCGCTGATGCAAACCTCGATAAGGTGTTGTCTGCTTACAACAAGCACAGAGATAAGCCTATCAGGGAACAGGCAGAGAACATCATCCATCTCTTCACCCTTACCAACCTCGGTGCTCCTGCTGCTTTCAAGTACTTCGATACAACCATCGATAGGAAGAGATACACCTCTACCAAAGAAGTGCTCGATGCTACCCTCATCCATCAGTCTATCACTGGACTCTACGAGACTAGGATCGATCTCTCACAGCTCGGTGGTGATTCAAGGGCTGATCCTAAGAAGAAGAGGAAGGTTGGAGACGACGGAGGTGGCGGTACAGGAGGGGGTGGGTCCGCTGAGTATGTCAGGGCGTTGTTCGACTTCAATGGAAACGACGAGGAAGATCTGCCTTTTAAAAAGGGAGATATTCTCAGGATCAGAGATAAGCCGGAAGAACAATGGTGGAACGCTGAAGACTCTGAAGGTAAGAGAGGTATGATTCTTGTCCCCTACGTCGAGAAGTATTCGGGTGACTATAAAGACCACGATGGAGATTATAAGGACCACGATATAGATTATAAGGATGATGATGATAAGAGCGGAATGACCGATGCAGAGTACGTCAGGATTCATGAGAAACTTGACATCTACACGTTTAAGAAACAGTTTTTCAACAACAAAAAATCTGTTAGTCACCGCTGTTACGTGCTGTTCGAATTGAAACGCAGAGGTGAGAGGAGAGCCTGCTTTTGGGGCTATGCCGTCAACAAGCCGCAAAGCGGCACAGAAAGGGGCATTCACGCGGAGATATTTAGCATTAGAAAGGTCGAGGAATACCTTCGGGATAATCCCGGGCAATTCACTATCAATTGGTACTCTTCATGGTCCCCGTGTGCAGATTGCGCTGAAAAGATACTGGAGTGGTATAATCAAGAACTCAGAGGAAACGGTCACACCCTCAAGATTTGGGCTTGCAAGCTTTACTACGAGAAAAATGCAAGGAACCAGATCGGCCTCTGGAACTTGCGCGACAACGGCGTGGGGTTGAATGTGATGGTGTCGGAGCATTACCAGTGCTGCCGGAAGATATTCATTCAGTCGTCACATAATCAATTGAACGAGAATAGGTGGCTCGAAAAAACCCTGAAGCGGGCCGAGAAGTGGAGGAGTGAACTCTCGATAATGATCCAGGTTAAAATACTGCATACTACCAAATCTCCGGCGGTGGGACCGAAGAAGAAGCGCAAGGTGGGGACCATGACTAATCTCTCAGATATAATCGAGAAGGAAACAGGAAAGCAACTGGTCATCCAAGAATCGATTTTGATGCTTCCCGAAGAAGTCGAAGAAGTTATAGGAAATAAGCCCGAGTCTGACATACTGGTTCACACAGCGTACGATGAAAGTACGGACGAGAATGTCATGTTGCTGACATCGGACGCACCTGAATACAAGCCTTGGGCTCTGGTCATACAAGATAGTAACGGAGAAAATAAGATTAAAATGCTTTCAGGTGGCTCCCCAAAGAAGAAACGCAAGGTTTGAGGATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGGAGACCTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGAAACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTGGTCTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGGATCCATATGAAGATGAAGATGAAATATTTGGTGTGTCAAATAAAAAGCTTGTGTGCTTAAGTTTGTGTTTTTTTCTTGGCTTGTTGTGTTATGAATTTGTGGCTTTTTCTAATATTAAATGAATGTAAGATCACATTATAATGAATAAACAAATGTTTCTATAATCCATTGTGAATGTTTTGTTGGATCTCTTCTGCAGCATATAACTACTGTATGTGCTATGGTATGGACTATGGAATATGATTAAAGATAAG

Seq ID No. 2: nCas9-PmCDA1 nuclease-cytosine deaminase fusion protein expression frame amino acid sequence

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSRADPKKKRKVGDDGGGGTGGGGSAEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWNAEDSEGKRGMILVPYVEKYSGDYKDHDGDYKDHDIDYKDDDDKSGMTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKWRSELSIMIQVKILHTTKSPAVGPKKKRKVGTMTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV。

Sequence listing

<110> university of electronic technology

<120> plant genome directed base editing framework vector and application thereof

<130> 20180001

<141> 2018-11-23

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8556

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cgcgcctgca gtgcagcgtg acccggtcgt gcccctctct tgagataatg agcattgcat 60

gtctaagtta taaaaaatta ccacatattt tttttgtcac acttgtttga agtgcagttt 120

atctatcttt atacatatat ttaaacttta ctctacgaat aatataatct atagtactac 180

aataatatca gtgttttaga gaatcatata aatgaacagt tagacatggt ctaaaggaca 240

attgagtatt ttgacaacag gactctacag ttttatcttt ttagtgtgca tgtgttctcc 300

tttttttttg caaatagctt cacctatata atacttcatc cattttatta gtacatccat 360

ttagggttta gggttaatgg tttttataga ctaatttttt tagtacatct attttattct 420

attttagcct ctaaattaag aaaactaaaa ctctatttta gtttttttat ttaataattt 480

agatataaaa tagaataaaa taaagtgact aaaaattaaa caaataccct ttaagaaatt 540

aaaaaaacta aggaaacatt tttcttgttt cgagtagata atgccagcct gttaaacgcc 600

gtcgacgagt ctaacggaca ccaaccagcg aaccagcagc gtcgcgtcgg gccaagcgaa 660

gcagacggca cggcatctct gtcgctgcct ctggacccct ctcgagagtt ccgctccacc 720

gttggacttg ctccgctgtc ggcatccaga aattgcgtgg cggagcggca gacgtgagcc 780

ggcacggcag gcggcctcct cctcctctca cggcaccggc agctacgggg gattcctttc 840

ccaccgctcc ttcgctttcc cttcctcgcc cgccgtaata aatagacacc ccctccacac 900

cctctttccc caacctcgtg ttgttcggag cgcacacaca cacaaccaga actcccccaa 960

atccacccgt cggcacctcc gcttcaaggt acgccgctcg tcctcccccc ccccccctct 1020

ctaccttctc aagatcggcg ttccggtcca tggttagggc ccggtagttc tacttctgtt 1080

catgtttgtg ttagatccgt gtttgtgtta gatccgtgct actagcgttc gtacacggat 1140

gcgacctgta cgtcagacac gttctgattg ctaacttgcc agtgtttctc tttggggaat 1200

cctgggatgg ctctagccgt tccgcagacg ggatcgattt catgattttt tttgtttcgt 1260

tgcatagggt ttggtttgcc cttttccttt atttcaatat atgccgtgca cttgtttgtc 1320

gggtcatctt ttcatgcttt tttttgtctt ggttgtgatg atgtggtctg gttgggcggt 1380

cgttcaagat cggagtagaa ttaattctgt ttcaaactac ctggtggatt tattaatttt 1440

ggatctgtat gtgtgtgcca tacatattca tagttacgaa ttgaagatga tggatggaaa 1500

tatcgatcta ggataggtat acatgttgat gcgggtttta ctgatgcata tacagagatg 1560

ctttttgttc gcttggttgt gatgatgtgg tgtggttggg cggtcgttca ttcgttcaag 1620

atcggagtag aatactgttt caaactacct ggtgtattta ttaattttgg aactgtatgt 1680

gtgtgtcata catcttcata gttacgagtt taagatggat ggaaatatcg atctaggata 1740

ggtatacatg ttgatgtggg ttttactgat gcatatacat gatggcatat gcagcatcta 1800

ttcatatgct ctaaccttga gtacctatct attataataa acaagtatgt tttataatta 1860

ttttgatctt gatatacttg gatgatggca tatgcagcag ctatatgtgg atttttttag 1920

ccctgccttc atacgctatt tatttgcttg gtactgtttc ttttgtcgat gctcaccctg 1980

ttgtttggtg ttacttctgc agcctgcagg atggataaga agtactctat cggactcgct 2040

atcggaacta actctgtggg atgggctgtg atcaccgatg agtacaaggt gccatctaag 2100

aagttcaagg ttctcggaaa caccgatagg cactctatca agaaaaacct tatcggtgct 2160

ctcctcttcg attctggtga aactgctgag gctaccagac tcaagagaac cgctagaaga 2220

aggtacacca gaagaaagaa caggatctgc tacctccaag agatcttctc taacgagatg 2280

gctaaagtgg atgattcatt cttccacagg ctcgaagagt cattcctcgt ggaagaagat 2340

aagaagcacg agaggcaccc tatcttcgga aacatcgttg atgaggtggc ataccacgag 2400

aagtacccta ctatctacca cctcagaaag aagctcgttg attctactga taaggctgat 2460

ctcaggctca tctacctcgc tctcgctcac atgatcaagt tcagaggaca cttcctcatc 2520

gagggtgatc tcaaccctga taactctgat gtggataagt tgttcatcca gctcgtgcag 2580

acctacaacc agcttttcga agagaaccct atcaacgctt caggtgtgga tgctaaggct 2640

atcctctctg ctaggctctc taagtcaaga aggcttgaga acctcattgc tcagctccct 2700

ggtgagaaga agaacggact tttcggaaac ttgatcgctc tctctctcgg actcacccct 2760

aacttcaagt ctaacttcga tctcgctgag gatgcaaagc tccagctctc aaaggatacc 2820

tacgatgatg atctcgataa cctcctcgct cagatcggag atcagtacgc tgatttgttc 2880

ctcgctgcta agaacctctc tgatgctatc ctcctcagtg atatcctcag agtgaacacc 2940

gagatcacca aggctccact ctcagcttct atgatcaaga gatacgatga gcaccaccag 3000

gatctcacac ttctcaaggc tcttgttaga cagcagctcc cagagaagta caaagagatt 3060

ttcttcgatc agtctaagaa cggatacgct ggttacatcg atggtggtgc atctcaagaa 3120

gagttctaca agttcatcaa gcctatcctc gagaagatgg atggaaccga ggaactcctc 3180

gtgaagctca atagagagga tcttctcaga aagcagagga ccttcgataa cggatctatc 3240

cctcatcaga tccacctcgg agagttgcac gctatcctta gaaggcaaga ggatttctac 3300

ccattcctca aggataacag ggaaaagatt gagaagattc tcaccttcag aatcccttac 3360

tacgtgggac ctctcgctag aggaaactca agattcgctt ggatgaccag aaagtctgag 3420

gaaaccatca ccccttggaa cttcgaagag gtggtggata agggtgctag tgctcagtct 3480

ttcatcgaga ggatgaccaa cttcgataag aaccttccaa acgagaaggt gctccctaag 3540

cactctttgc tctacgagta cttcaccgtg tacaacgagt tgaccaaggt taagtacgtg 3600

accgagggaa tgaggaagcc tgcttttttg tcaggtgagc aaaagaaggc tatcgttgat 3660

ctcttgttca agaccaacag aaaggtgacc gtgaagcagc tcaaagagga ttacttcaag 3720

aaaatcgagt gcttcgattc agttgagatt tctggtgttg aggataggtt caacgcatct 3780

ctcggaacct accacgatct cctcaagatc attaaggata aggatttctt ggataacgag 3840

gaaaacgagg atatcttgga ggatatcgtt cttaccctca ccctctttga agatagagag 3900

atgattgaag aaaggctcaa gacctacgct catctcttcg atgataaggt gatgaagcag 3960

ttgaagagaa gaagatacac tggttgggga aggctctcaa gaaagctcat taacggaatc 4020

agggataagc agtctggaaa gacaatcctt gatttcctca agtctgatgg attcgctaac 4080

agaaacttca tgcagctcat ccacgatgat tctctcacct ttaaagagga tatccagaag 4140

gctcaggttt caggacaggg tgatagtctc catgagcata tcgctaacct cgctggatct 4200

cctgcaatca agaagggaat cctccagact gtgaaggttg tggatgagtt ggtgaaggtg 4260

atgggaaggc ataagcctga gaacatcgtg atcgaaatgg ctagagagaa ccagaccact 4320

cagaagggac agaagaactc tagggaaagg atgaagagga tcgaggaagg tatcaaagag 4380

cttggatctc agatcctcaa agagcaccct gttgagaaca ctcagctcca gaatgagaag 4440

ctctacctct actacctcca gaacggaagg gatatgtatg tggatcaaga gttggatatc 4500

aacaggctct ctgattacga tgttgatcat atcgtgccac agtcattctt gaaggatgat 4560

tctatcgata acaaggtgct caccaggtct gataagaaca ggggtaagag tgataacgtg 4620

ccaagtgaag aggttgtgaa gaaaatgaag aactattgga ggcagctcct caacgctaag 4680

ctcatcactc agagaaagtt cgataacttg actaaggctg agaggggagg actctctgaa 4740

ttggataagg caggattcat caagaggcag cttgtggaaa ccaggcagat cactaagcac 4800

gttgcacaga tcctcgattc taggatgaac accaagtacg atgagaacga taagttgatc 4860

agggaagtga aggttatcac cctcaagtca aagctcgtgt ctgatttcag aaaggatttc 4920

caattctaca aggtgaggga aatcaacaac taccaccacg ctcacgatgc ttaccttaac 4980

gctgttgttg gaaccgctct catcaagaag tatcctaagc tcgagtcaga gttcgtgtac 5040

ggtgattaca aggtgtacga tgtgaggaag atgatcgcta agtctgagca agagatcgga 5100

aaggctaccg ctaagtattt cttctactct aacatcatga atttcttcaa gaccgagatt 5160

accctcgcta acggtgagat cagaaagagg ccactcatcg agacaaacgg tgaaacaggt 5220

gagatcgtgt gggataaggg aagggatttc gctaccgtta gaaaggtgct ctctatgcca 5280

caggtgaaca tcgttaagaa aaccgaggtg cagaccggtg gattctctaa agagtctatc 5340

ctccctaaga ggaactctga taagctcatt gctaggaaga aggattggga ccctaagaaa 5400

tacggtggtt tcgattctcc taccgtggct tactctgttc tcgttgtggc taaggttgag 5460

aagggaaaga gtaagaagct caagtctgtt aaggaacttc tcggaatcac tatcatggaa 5520

aggtcatctt tcgagaagaa cccaatcgat ttcctcgagg ctaagggata caaagaggtt 5580

aagaaggatc tcatcatcaa gctcccaaag tactcactct tcgaactcga gaacggtaga 5640

aagaggatgc tcgcttctgc tggtgagctt caaaagggaa acgagcttgc tctcccatct 5700

aagtacgtta actttcttta cctcgcttct cactacgaga agttgaaggg atctccagaa 5760

gataacgagc agaagcaact tttcgttgag cagcacaagc actacttgga tgagatcatc 5820

gagcagatct ctgagttctc taaaagggtg atcctcgctg atgcaaacct cgataaggtg 5880

ttgtctgctt acaacaagca cagagataag cctatcaggg aacaggcaga gaacatcatc 5940

catctcttca cccttaccaa cctcggtgct cctgctgctt tcaagtactt cgatacaacc 6000

atcgatagga agagatacac ctctaccaaa gaagtgctcg atgctaccct catccatcag 6060

tctatcactg gactctacga gactaggatc gatctctcac agctcggtgg tgattcaagg 6120

gctgatccta agaagaagag gaaggttgga gacgacggag gtggcggtac aggagggggt 6180

gggtccgctg agtatgtcag ggcgttgttc gacttcaatg gaaacgacga ggaagatctg 6240

ccttttaaaa agggagatat tctcaggatc agagataagc cggaagaaca atggtggaac 6300

gctgaagact ctgaaggtaa gagaggtatg attcttgtcc cctacgtcga gaagtattcg 6360

ggtgactata aagaccacga tggagattat aaggaccacg atatagatta taaggatgat 6420

gatgataaga gcggaatgac cgatgcagag tacgtcagga ttcatgagaa acttgacatc 6480

tacacgttta agaaacagtt tttcaacaac aaaaaatctg ttagtcaccg ctgttacgtg 6540

ctgttcgaat tgaaacgcag aggtgagagg agagcctgct tttggggcta tgccgtcaac 6600

aagccgcaaa gcggcacaga aaggggcatt cacgcggaga tatttagcat tagaaaggtc 6660

gaggaatacc ttcgggataa tcccgggcaa ttcactatca attggtactc ttcatggtcc 6720

ccgtgtgcag attgcgctga aaagatactg gagtggtata atcaagaact cagaggaaac 6780

ggtcacaccc tcaagatttg ggcttgcaag ctttactacg agaaaaatgc aaggaaccag 6840

atcggcctct ggaacttgcg cgacaacggc gtggggttga atgtgatggt gtcggagcat 6900

taccagtgct gccggaagat attcattcag tcgtcacata atcaattgaa cgagaatagg 6960

tggctcgaaa aaaccctgaa gcgggccgag aagtggagga gtgaactctc gataatgatc 7020

caggttaaaa tactgcatac taccaaatct ccggcggtgg gaccgaagaa gaagcgcaag 7080

gtggggacca tgactaatct ctcagatata atcgagaagg aaacaggaaa gcaactggtc 7140

atccaagaat cgattttgat gcttcccgaa gaagtcgaag aagttatagg aaataagccc 7200

gagtctgaca tactggttca cacagcgtac gatgaaagta cggacgagaa tgtcatgttg 7260

ctgacatcgg acgcacctga atacaagcct tgggctctgg tcatacaaga tagtaacgga 7320

gaaaataaga ttaaaatgct ttcaggtggc tccccaaaga agaaacgcaa ggtttgagga 7380

tctaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaacaaagca 7440

ccagtggtct agtggtagaa tagtaccctg ccacggtaca gacccgggtt cgattcccgg 7500

ctggtgcagg agaccttata ttccccagaa catcaggtta atggcgtttt tgatgtcatt 7560

ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gaaaccggca cactggccat 7620

atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg 7680

ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcgcccggg 7740

cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct ctcttttata 7800

ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa gagccgttca 7860

tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc cagcgttcgg 7920

cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag atattgacat 7980

catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa tacgctgctt 8040

catagcatac ctctttttga catacttcgg gtatacatat cagtatatat tcttataccg 8100

caaaaatcag cgcgcaaata cgcatactgt tatctggctt ggtctcagtt ttagagctag 8160

aaatagcaag ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg 8220

tgcaacaaag caccagtggt ctagtggtag aatagtaccc tgccacggta cagacccggg 8280

ttcgattccc ggctggtgca ggatccatat gaagatgaag atgaaatatt tggtgtgtca 8340

aataaaaagc ttgtgtgctt aagtttgtgt ttttttcttg gcttgttgtg ttatgaattt 8400

gtggcttttt ctaatattaa atgaatgtaa gatcacatta taatgaataa acaaatgttt 8460

ctataatcca ttgtgaatgt tttgttggat ctcttctgca gcatataact actgtatgtg 8520

ctatggtatg gactatggaa tatgattaaa gataag 8556

<210> 2

<211> 1788

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg

965 970 975

Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val

980 985 990

Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

995 1000 1005

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys

1010 1015 1020

Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser

1025 1030 1035 1040

Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu

1045 1050 1055

Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile

1060 1065 1070

Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser

1075 1080 1085

Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly

1090 1095 1100

Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile

1105 1110 1115 1120

Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser

1125 1130 1135

Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly

1140 1145 1150

Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile

1155 1160 1165

Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala

1170 1175 1180

Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys

1185 1190 1195 1200

Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser

1205 1210 1215

Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr

1220 1225 1230

Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1235 1240 1245

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His

1250 1255 1260

Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val

1265 1270 1275 1280

Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys

1285 1290 1295

His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu

1300 1305 1310

Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp

1315 1320 1325

Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp

1330 1335 1340

Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile

1345 1350 1355 1360

Asp Leu Ser Gln Leu Gly Gly Asp Ser Arg Ala Asp Pro Lys Lys Lys

1365 1370 1375

Arg Lys Val Gly Asp Asp Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser

1380 1385 1390

Ala Glu Tyr Val Arg Ala Leu Phe Asp Phe Asn Gly Asn Asp Glu Glu

1395 1400 1405

Asp Leu Pro Phe Lys Lys Gly Asp Ile Leu Arg Ile Arg Asp Lys Pro

1410 1415 1420

Glu Glu Gln Trp Trp Asn Ala Glu Asp Ser Glu Gly Lys Arg Gly Met

1425 1430 1435 1440

Ile Leu Val Pro Tyr Val Glu Lys Tyr Ser Gly Asp Tyr Lys Asp His

1445 1450 1455

Asp Gly Asp Tyr Lys Asp His Asp Ile Asp Tyr Lys Asp Asp Asp Asp

1460 1465 1470

Lys Ser Gly Met Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu

1475 1480 1485

Asp Ile Tyr Thr Phe Lys Lys Gln Phe Phe Asn Asn Lys Lys Ser Val

1490 1495 1500

Ser His Arg Cys Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg

1505 1510 1515 1520

Arg Ala Cys Phe Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr

1525 1530 1535

Glu Arg Gly Ile His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu

1540 1545 1550

Tyr Leu Arg Asp Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser

1555 1560 1565

Trp Ser Pro Cys Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn

1570 1575 1580

Gln Glu Leu Arg Gly Asn Gly His Thr Leu Lys Ile Trp Ala Cys Lys

1585 1590 1595 1600

Leu Tyr Tyr Glu Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu

1605 1610 1615

Arg Asp Asn Gly Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln

1620 1625 1630

Cys Cys Arg Lys Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu

1635 1640 1645

Asn Arg Trp Leu Glu Lys Thr Leu Lys Arg Ala Glu Lys Trp Arg Ser

1650 1655 1660

Glu Leu Ser Ile Met Ile Gln Val Lys Ile Leu His Thr Thr Lys Ser

1665 1670 1675 1680

Pro Ala Val Gly Pro Lys Lys Lys Arg Lys Val Gly Thr Met Thr Asn

1685 1690 1695

Leu Ser Asp Ile Ile Glu Lys Glu Thr Gly Lys Gln Leu Val Ile Gln

1700 1705 1710

Glu Ser Ile Leu Met Leu Pro Glu Glu Val Glu Glu Val Ile Gly Asn

1715 1720 1725

Lys Pro Glu Ser Asp Ile Leu Val His Thr Ala Tyr Asp Glu Ser Thr

1730 1735 1740

Asp Glu Asn Val Met Leu Leu Thr Ser Asp Ala Pro Glu Tyr Lys Pro

1745 1750 1755 1760

Trp Ala Leu Val Ile Gln Asp Ser Asn Gly Glu Asn Lys Ile Lys Met

1765 1770 1775

Leu Ser Gly Gly Ser Pro Lys Lys Lys Arg Lys Val

1780 1785

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgacatccgc aagtaccagg 20

<210> 4

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

agtacactgt ttccccgtat gt 22

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gctgctggtg agtgctgat 19

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

acccattggg agtgtcttgc 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaccagccag cgtctggcgc 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcagctggct gagggtgcat 20

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agccagctgc ttacaaaac 19

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgcagaccag ccagcgtctg gcgc 24

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aaacgcgcca gacgctggct ggtc 24

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgcagcagct ggctgagggt gcat 24

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

aaacatgcac cctcagccag ctgc 24

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgcaagccag ctgcttacaa aac 23

<210> 15

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

aaacgttttg taagcagctg gct 23

Claims (10)

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.