CN110607320A - A plant genome directed base editing backbone vector and its application - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to a plant genome-directed base editing backbone carrier and its application. The technical problem to be solved by the present invention is to improve the efficiency of targeted base editing in plant cell genomes and expand the base editing window. The technical solution of the present invention to solve the technical problem is to provide a plant genome-directed base editing backbone carrier, which is driven by a Pol II type promoter to express the nCas9-PmCDA1 nuclease-cytidine deaminase fusion protein expression unit and synthetic guide RNA (sgRNA) Transcription of the core unit composed of two core regions of the transcription expression unit. The single transcription unit directional base editing backbone carrier of the present invention can effectively realize the simple, fast and efficient directional editing of cytosine base (C) into thymine base (T), and is a kind of effective realization of plant genome base directional editing. Molecular tools for editing.

Description

A plant genome directed base editing backbone vector and its application

technical field

The invention belongs to the field of plant genetic engineering, and relates to a plant genome-directed base editing backbone carrier and an application thereof.

Background technique

Targeted genome modification has always been a frontier and hot spot in biological research. Through precise targeted modification of specific regions of the genome: on the one hand, it can precisely mutate the target sequence, obtain mutation materials, and clearly identify the function of the target gene; on the other hand, it can Precise replacement or insertion of the target sequence minimizes the uncertainty of expression and inheritance caused by the random introduction of foreign genes.

In 2012, researchers proved for the first time that CRISPR-Cas (Clustered regularly interspacedshort palindromic repeats-CRISPR associated protein) can achieve sequence-specific DNA double-strand shearing, and then the CRISPR-Cas9 system was used in cynomolgus monkeys, zebrafish, mice, In human cell lines, Arabidopsis, rice and other animal and plant systems, RNA-guided intracellular genome-directed editing has been realized. In this genome-directed editing system, the Cas protein, guided by the guide RNA, recognizes and cuts specific DNA sequences to generate DNA double strand breaks (double strand breaks, DSBs), and then realizes the DNA sequence orientation of the target site based on the endogenous DNA repair system of the cell. edit. Currently known eukaryotic DNA repair systems can be divided into two categories: "homologous recombination" (homologous recombination, HR) repair; "nonhomologous end joining" (nonhomologous end joining, NHEJ) repair. HR uses homologous sequences as templates to accurately repair damaged DNA regions; while NHEJ does not require the existence of homologous sequences, and directly joins the broken ends formed by DNA damage, and often introduces different degrees of sequence variation when repairing .

Although the CRISPR-Cas genome editing tool can effectively realize the directional editing of the target genome sequence, the editing events based on the NHEJ repair pathway mainly introduce base insertion or deletion mutations randomly at the target modification site, while the editing events based on the HR repair pathway can The sequence replacement of the target modification site is accurately carried out based on the donor template DNA, but its frequency and efficiency are much lower than the editing events mediated by the NHEJ repair pathway, which greatly limits the basic research and development related to precise base editing of CRISPR-Cas genome editing tools. Effective application of applied practice.

In order to improve the efficiency of precise editing of specific bases at target sites in the genome and effectively achieve precise single-base substitution editing at target sites, researchers based on the CRISPR-Cas genome editing tool combined specific base deaminases with dCas9, nCas9 or The fusion of your Cas12a achieves the precise substitution and editing of specific bases at the target site of the genome (for example: base C is replaced by base T; base A is replaced by base G), this new genome editing tool is called Base editor (baseeditor, BE). Targeted base editing technology, which can effectively replace and edit specific single bases at genome target sites, is a useful supplement to CRISPR-Cas genome editing technology, and was named one of the top ten annual scientific breakthroughs in the world by Science magazine in 2017 , highlighting the important potential of this technology in basic research and applied practice.

The directional base editing tool based on the CRISPR-Cas system has effectively expanded the application range of the CRISPR-Cas system, showing its broad application prospects. However, the existing directional base editing tools generally have the problems of low editing efficiency and limited editing window, especially in the application practice of plant genome editing. An enhanced plant-directed base editing tool for the base editing window, in order to effectively expand the positive application of the CRISPR-Cas system-based directed base editing technology in plant genome function research and breeding practice.

Contents of the invention

The technical problem to be solved by the present invention is to improve the efficiency of targeted base editing in plant cell genomes and expand the base editing window.

The technical solution of the present invention to solve the technical problem is to provide a plant genome-directed base editing backbone vector. The backbone vector contains a core unit consisting of two core regions, an nCas9-PmCDA1 nuclease-cytidine deaminase fusion protein expression unit and a synthetic guide RNA (sgRNA) transcriptional expression unit, which is driven by a PolⅡ-type promoter transcription;

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

Wherein, the functional unit of the PmCDA1 cytosine deaminase coding region described in the above-mentioned backbone vector includes GGGS linker, SH3 linker, PmCDA1 coding region, NLS signal peptide, UGI coding region, SGGS linker, NLS signal sequence from N-terminal to C-terminal peptide.

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

a. The amino acid sequence encoded by the nCas9 ORF coding frame of the nCas9 nuclease protein D10A mutant is shown in amino acids 1 to 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 in amino acids 1383 to 1788 in Seq ID No.2.

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

Wherein, the number of sgRNA clones and transcription units described in the above-mentioned backbone vector is 1-6.

Wherein, the nucleotide sequence of the sgRNA clone and the transcription unit sgRNA cloning scaffold described in the above-mentioned backbone vector is shown in the 7432bp to the 8300bp in Seq ID No.1.

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

a. The nucleotide sequence encoded by the nCas9 ORF coding frame of nCas9 nuclease protein D10A mutant is shown in 2011bp to 6156bp in Seq ID No.1;

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

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

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

e. The Pol II type promoter is the maize pZmUbi1 promoter pZmUbi1, and its nucleotide sequence is shown in the 1st bp to the 2008th bp in Seq ID No.1.

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

Based on the above-mentioned backbone vector, the present invention also provides a method for preparing a recombinant expression vector for targeted base editing of a specific cytosine base at a target site in a plant genome. The method comprises the steps of:

a. Identify the target DNA region of a specific biological genome, analyze the region with the characteristics of PAM (PAM full name protospaceradjacent motif sequence, adjacent motif of candidate recognition site), and select the 15-30bp DNA sequence adjacent to the 5' end of the PAM structure as the specific target sequence;

b. According to the selected specific target sequence, synthesize the forward oligonucleotide chain with 5'-CGGA-N _X -3' characteristics and the reverse oligonucleotide chain with 5'-AAAC-N _X -3' characteristics Nucleotide chain, N represents any one of A, G, C, T, X is an integer, and 14≤X≤30, wherein the N _X in the forward oligonucleotide chain and the reverse oligonucleotide _NX in the nucleotide has reverse complementary characteristics; the complementary oligonucleotide double-stranded fragment is obtained by annealing;

c. Mix the plant genome-directed base editing backbone carrier according to any one of claims 1 to 9 with the complementary oligonucleotide double-stranded fragment obtained in step b, and add BsaI endonuclease and T4 DNA connection to the reaction system at the same time Enzyme, set up restriction restriction-ligation cycle reaction, obtain the recombinant expression vector for targeted base editing.

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

Preferably, 18≤X≤21 in step b.

Preferably, in practical operation, the fusion PCR amplification strategy can be applied in step c to obtain the tandem amplification products of multiple sgRNA transcription units separated by tRNA sequences, and the cycle reaction of "BsaI digestion-T4 DNA ligase connection" In this way, the BsaI-ccdB-BsaI unit is replaced, and the multiple sgRNA transcription unit is cloned into the sgRNA clone and transcription unit to obtain a recombinant expression vector capable of specific and directional base editing for multiple target sites.

The beneficial effect of the present invention is that the present invention is driven by a promoter to drive the two core regions of the nCas9-PmCDA1 fusion protein and the synthetic guide RNA (sgRNA) transcription expression unit, constituting the core unit of the single transcription unit directed base editing backbone vector . Using the directional base editing backbone vector containing this core unit, the simple, fast and efficient directional editing of cytosine base (C) into thymine base (T) can be effectively realized for the target sequence of plant genome. Compared with the currently used plant base editing tools, the present invention improves the base editing efficiency, expands the base editing window, and promotes the effective application of the directional base editing strategy in the directional editing of plant genomes. It is an effective implementation Molecular tools for base-directed editing of plant genomes have promising application prospects.

Description of drawings

Fig. 1. The core unit structure and working schematic diagram of the plant STU nCas9-PmCDA1 single transcription unit directed base editing backbone vector in the present invention.

Figure 2. Transient transformation of rice protoplasts based on STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, and STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vectors in the present invention, based on the target of Illumina high-throughput sequencing Analysis of locus cytosine-directed editing efficiency. Wherein, nCas9-PmCDA1 represents the plant STU nCas9-PmCDA1 single transcription unit directional base editing backbone carrier in the present invention, and nCas9-rApobec1 is a control group (according to references reported (Komor AC, Kim YB, Packer MS, Zuris JA, LiuDR. 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature, 533(7603):420-424.), replace rApobec1 cytosine deaminase with the single transcription of the plant STU nCas9-PmCDA1 constructed in the present invention PmCDA1 unit in a unit-directed base editing backbone vector).

Figure 3 is based on the transient transformation of rice protoplasts with the STU nCas9-PmCDA1-OsCDC48-sgRNA01 recombinant expression vector in the present invention, and performing Illumina high-throughput sequencing, and replacing cytosine base sites at different positions at specific editing sites with thymine bases Edit efficiency analysis. Among them, nCas9-PmCDA1 and nCas9-rApobec1 are as illustrated in Figure 2.

Figure 4 is based on the transient transformation of rice protoplasts with the STU nCas9-PmCDA1-OsROC5-gRNA04 recombinant expression vector in the present invention, and performing Illumina high-throughput sequencing, and replacing cytosine base sites at different positions at specific editing sites with thymine bases Edit efficiency analysis. Among them, nCas9-PmCDA1 and nCas9-rApobec1 are as illustrated in Figure 2.

Figure 5 is based on the transient transformation of rice protoplasts with the STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vector in the present invention, and performing Illumina high-throughput sequencing, and replacing cytosine base sites at different positions at specific editing sites with thymine bases Edit efficiency analysis. Among them, nCas9-PmCDA1 and nCas9-rApobec1 are as illustrated in Figure 2.

Figure 6 is based on the STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vectors in the present invention to perform Agrobacterium-mediated genetic transformation of rice, and extract rice to transform and regenerate seedlings Genomic DNA was subjected to PCR amplification and Sanger sequencing analysis, and the result of target site cytosine-directed editing efficiency analysis.

Detailed ways

Based on the CRISPR-Cas9 single transcription system and PmCDA1 cytosine deaminase, the present invention constructs a plant genome-directed base editing backbone vector through strategies such as codon optimization in the coding region and multiple assembly of functional units. called plant STU nCas9-PmCDA1 single transcription unit directed base editing backbone vector),

The plant genome-directed base editing backbone vector of the present invention comprises a core unit consisting of two core regions, an nCas9-PmCDA1 nuclease-cytidine deaminase fusion protein expression unit and a synthetic guide RNA (sgRNA) transcription and expression unit, the core unit Transcription driven by a Pol II promoter;

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

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

Wherein, the functional unit of the PmCDA1 cytosine deaminase coding region described in the above-mentioned backbone vector includes GGGS linker, SH3 linker, PmCDA1 coding region, NLS signal peptide, UGI coding region, SGGS linker, NLS signal sequence from N-terminal to C-terminal peptide.

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

a. The amino acid sequence encoded by the nCas9 ORF coding frame of the nCas9 nuclease protein D10A mutant is shown in amino acids 1 to 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 in amino acids 1383 to 1788 in Seq ID No.2. The above two components are connected to form the nCas9-PmCDA1 nuclease-cytidine deaminase fusion protein expression cassette, and the amino acid sequence is shown in Seq ID No.2 in the sequence list.

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

Wherein, the number of sgRNA clones and transcription units described in the above-mentioned backbone vector is 1-6.

Wherein, the nucleotide sequence of the sgRNA clone and the transcription unit sgRNA cloning scaffold described in the above-mentioned backbone vector is shown in the 7432bp to the 8300bp in Seq ID No.1.

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

a. The nucleotide sequence encoded by the nCas9 ORF coding frame of nCas9 nuclease protein D10A mutant is shown in 2011bp to 6156bp in Seq ID No.1;

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

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

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

e. The Pol II type promoter is the maize pZmUbi1 promoter pZmUbi1, and its nucleotide sequence is shown in the 1st bp to the 2008th bp in Seq ID No.1.

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

The core unit of the present invention (pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloningscaffold-HSP T) can be aimed at specific transformation host organisms and experimental needs, and replace the promoter and terminator with any Pol II type promoter element (such as: promoter elements such as OsUb1, CaMV35S, AtUb10 commonly used in plants) and terminator elements (such as: terminator elements such as Nos T and 35s T commonly used in plants), and can be placed in any plant expression backbone vector ( Such as: pCambia, pBI, pMDC, pGreen and other vector series commonly used in plants) to achieve site-specific directional base editing.

In the present invention, based on the plant STU nCas9-PmCDA1 single transcription unit directional base editing backbone carrier, after completing the construction of specific plant genome site-specific STU nCas9-PmCDA1-sgRNA directional base editing recombinant expression vectors for transformation, in living cell conditions Under this condition, the PolⅡ promoter drives the "nCas9 ORF-PmCDA1-Poly A-sgRNAcloning scaffold" as an overall transcription unit to transcribe a single-stranded primary transcript. Under the action of endogenous tRNA processing factors, a single primary transcript was self-cleaved at two tRNA sites to obtain a complete nCas9 ORF-PmCDA1 nuclease-cytidine deaminase fusion protein expression frame mRNA (containing Poly A ) and sgRNA transcription unit. In the cell system, the nCas9-PmCDA1 nuclease-cytidine deaminase fusion protein expression cassette (including Poly A) is further translated to obtain the nCas9-PmCDA1 nuclease-cytidine deaminase fusion protein, which is combined with the existing sgRNA unit Combined to form a functional nCas9-PmCDA1-sgRNA complex unit for targeted editing of specific cytosine bases at the target site of the genome.

In the present invention, the complete sgRNA is formed by replacing the BsaI-ccdB-BsaI unit in the backbone carrier sgRNA clone and transcription unit with an 18-21bp RNA fragment capable of complementary binding to the target fragment, and the backbone RNA fragment is sequentially composed of The sgRNA, tracrRNA, and crRNA at the protospacer site are chimerized to form a functional RNA similar to a hairpin structure, and the backbone RNA fragment can be combined with the Cas9 nuclease.

For the specific target gene, after determining the sgRNA site (5'-N _X -NGG-3'; N represents any of A, G, C, T, X is an integer, and 14≤X≤30 (18 , 19, 20, and 21 are commonly used values)), according to the STU nCas9-PmCDA1-sgRNA recombinant expression vector construction method provided in the invention, the designed sgRNA-specific target sequence (protospacer) "BsaI digestion-T4 DNA ligase connection "Circular reaction method, replace the BsaI-ccdB-BsaI unit and clone into the gRNA clone and transcription unit to obtain a specific functional STU nCas9-PmCDA1-sgRNA recombinant expression vector.

In the present invention, the BsaI-ccdB-BsaI unit is fused at the end of the sgRNA clone transcription framework unit, which acts as a multi-cloning site to digest the CRISPR/Cas9 single transcription unit backbone vector so as to clone the target gRAN-specific target sequence (protospacer) . The key content of the present invention can be effectively realized by replacing the BsaI-ccdB-BsaI unit with a restriction endonuclease that can introduce a nick on the backbone vector of the present invention, and modifying the sgRAN-specific target sequence cloning site accordingly.

In the process of constructing plant genome site-specific STU nCas9-PmCDA1-sgRNA-directed base editing recombinant expression vector, the recombinant clone containing the correct Cas9-gRNA expression vector can be screened by transforming Escherichia coli and bacterial screening pressure, and colony PCR can be used , Plasmid digestion, sequence determination and other methods were identified to clearly obtain the target plant genome site-specific STU nCas9-PmCDA1-sgRNA directional base editing recombinant expression vector.

Using the fusion PCR amplification strategy, multiple sgRNA transcription units separated by tRNA sequences can be amplified in series, and the BsaI-ccdB-BsaI unit can be replaced by the circular reaction method of "BsaI digestion-T4 DNA ligase ligation". This multiple sgRNA transcription unit is cloned into the sgRNA clone and transcription unit to obtain a STU nCas9-PmCDA1-sgRNA1-sgRNA2-…-sgRNAx recombinant expression vector that can be specifically modified for multiple target sites (see Figure 1). Preferably, the number of sgRNA clones and transcription units described in the above-mentioned backbone vector is 1-6.

In the present invention, the site-specific STU nCas9-PmCDA1-sgRNA directional base-editing recombinant expression vector constructed according to the present invention can be transferred into plant cells through various transformation methods mediated by protoplasts, gene guns, and Agrobacterium, so that Transformed cells have both nCas9-PmCDA1 nuclease-CD fusion protein and sgRNA unit targeting specific genomic target sequences; under the joint action of nCas9-PmCDA1 nuclease-CD fusion protein and sgRNA unit, specific Genomic target sequence specific cytosine bases are edited (replace them with T (high probability), A (low probability), G (low probability)). When the STU nCas9-PmCDA1 single transcription unit directional base editing backbone vector of the present invention is applied in plants, resistance genes including kanamycin, hygromycin, basta, etc. can be used to screen plant transformants, and positive transformation Daughter cells or tissues (such as protoplasts or callus) are differentiated and regenerated to obtain regenerated plants containing targeted modification of the target site.

Example 1 Construction of Plant STU nCas9-PmCDA1 Single Transcription Unit Directed Base Editing Backbone Vector

The present invention designs a STU nCas9-PmCDA1 single transcription unit directional base editing backbone carrier for plant genome engineering, and its core unit is pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold from 5' to 3' direction -HSP T. Among them: pZmUbi1 is the maize pZmUbi1 promoter (the basic STU nCas9-PmCDA1 single transcription unit-directed base-editing backbone carrier scheme can be used to realize the replacement of different PolII-type promoters through AscI and SbfI double enzyme digestion); nCas9 ORF is the suppurative chain Cocci (Streptococcus pyogenes) nuclease protein D10A mutant coding frame (including C-terminal NLS signal peptide); region, NLS signal peptide, UGI coding region, SGGS linker, NLS signal peptide); Poly A is the poly A region; sgRNA cloning scaffold (abbreviated as sgRNA CS) is the sgRNA clone and transcription unit (from 5' end to 3' end Contains tRNA-Gly coding sequence, BsaI-ccdB-BsaI unit, sgRNA backbone coding sequence, tRNA-Gly coding sequence); HSP T is rice HSP terminator (basic STUnCas9-PmCDA1 single transcription unit can be digested by BamHI and SacI double enzymes A protocol for directed base editing backbone vectors to achieve the replacement of different terminators). The structure and working principle of the plant STU nCas9-PmCDA1 single transcription unit directed base editing backbone vector are shown in Figure 1.

Optionally, the backbone vector also includes: the left and right border sequences of T-DNA, and the "pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSP T" core unit is located at the left and right borders of the T-DNA between the left and right border sequences of T-DNA can also include the hygromycin resistance gene expression unit (consisting elements in turn are: 2×CaMV35S promoter-hygromycin phosphotransferase ORF-CaMV poly A; can pass AvrII, PacI double The scheme of enzyme-digesting basic CRISPR/Cas9 single transcription unit backbone vector to realize the replacement of ORF of different resistance genes) as a screening marker for plant transformants.

In order to realize the rapid and efficient construction of the specific genome target STU nCas9-PmCDA1-sgRNA directional base editing recombinant expression vector, the plant STU nCas9-PmCDA1 single transcription unit directional base editing backbone vector of the present invention is in the 5 The 637bp BsaI-ccdB-BsaI unit is integrated into the ' end. Based on this design strategy, in the subsequent construction process of the target STU nCas9-PmCDA1-sgRNA directional base editing recombinant expression vector, it is only necessary to mix the BsaI described in the present invention in the construction system. Plant STU nCas9-PmCDA1 single transcription unit directional base editing backbone vector, annealed specific target sequence complementary oligonucleotide double-stranded fragment, BsaI endonuclease and T4 DNA ligase, and set "37°C enzyme digestion -16°C The efficient construction of a specific Cas9-gRNA expression vector can be realized by connecting” cycle reaction. The vector can be constructed in a conventional way in the existing molecular cloning technology, and it should be noted that the above-mentioned element sequence is a unique part of the backbone plasmid vector, and it can also include some general structures of conventional vectors, which are not included in the present invention. Let me tell you more.

Based on Streptococcus pyogenes (Streptococcus pyogenes) Cas9 nuclease protein coding gene (Streptococcus pyogenes Cas9, SpCas9), the plant codon was optimized (the NLS signal was added at the 3' end), and the D10A mutation was introduced to artificially synthesize the nCas9 nuclease protein D10A mutant The complete ORF sequence of the coding frame (including the C-terminal NLS coding sequence), the nucleotide sequence is shown in 2011bp to 6156bp in Seq ID No.1 (the encoded amino acid sequence is shown in 1AA to 1382AA in Seq ID No.2 ). At the same time, according to the detection sequence of lamprey (Petromyzon marinus) cytosine deaminase (PmCDA1) (Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A.2016.Targetednucleotide editing using hybrid prokaryotic and vertebrate adaptive immunesystems.Science,353(6305).pii:aaf8729), design PmCDA1 cytosine deaminase expression unit coding frame (from N-terminal to C-terminal Contains GGGS linker, SH3 linker, PmCDA1 coding region, NLS signal peptide, UGI coding region, SGGS linker, NLS signal peptide in turn), plant codon optimization and artificial synthesis, nucleotide sequence as in Seq ID No.1 6157bp to 7374bp (the coded amino acid sequence is shown as 1383AA to 1788AA in Seq ID No.2). Further, another 3 basic units are obtained through artificial synthesis:

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

b. frag-B: sgRNA clone and transcription unit coding sequence, including tRNA-Gly coding sequence, BsaI-ccdB-BsaI unit, sgRNA backbone coding sequence, tRNA-Gly coding sequence from 5' end to 3' end: nucleoside The acid sequence is shown in the 7432bp to the 8300bp in Seq ID No.1);

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

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

The vector backbone 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 efficient genome editing in plants. Molecular Plant, 9( 7): 1088-1091.) and 6560bp assembly unit were subjected to SbfI and SacI double enzyme digestion, and the target fragment was recovered for connection and transformation. Colony PCR, plasmid restriction enzyme digestion, and DNA sequencing were performed on the screened positive clones to confirm that the 6560bp assembly unit was cloned into the downstream of the original pZmUbi1 of pGEL026, and the construction of the plant STU nCas9-PmCDA1 single transcription unit-directed base editing backbone vector was completed.

Example 2 High-throughput identification of rice endogenous gene cytosine-directed base editing efficiency based on STU nCas9-PmCDA1 system

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

For rice OsCDC48 (LOC_Os03g05730) and OsROC5 (LOC_Os02g45250) coding genes, the 5'-NGG-3'PAM site was searched, and the 20bp sequence upstream of the PAM was selected to design sgRNA (Table 1).

Table 1 Design, synthesis and detection information of rice endogenous gene sgRNAcrRNA

According to the designed sgRNA site nucleic acid sequence, artificially synthesize the corresponding forward and reverse oligonucleotide chains, the specific sequence is as follows (uppercase base sequence represents the designed site-specific guide sgRNA site; lowercase base sequence represents the site with Backbone vector complementary cohesive ends):

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.

Mix BE-OsCDC48-sgRNA01-F/R, BE-OsROC5-gRNA04-F/R, and BE-OsROC5-gRNA05-F/R in equal proportions, put them in a boiling water bath for 10 minutes, and then cool down and anneal naturally to form a doublet with sticky ends. stranded DNA as an insert for constructing recombinant vectors. Add the plant STU nCas9-PmCDA1 single transcription unit directed base editing backbone vector, sticky-end insert, BsaI endonuclease and T4 DNA ligase into a 200uL PCR tube, and set up 10 cycles of "37°C digestion-16°C ligation" After the reaction, the endonuclease and ligase were inactivated after being treated at 80°C, and the reaction product was taken for Escherichia coli transformation.

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

2. Rice protoplast transformation of rice endogenous gene STU nCas9-PmCDA1-sgRNA base editing recombinant expression vector

Isolate rice Nipponbare protoplasts, and transform rice protoplasts with STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, and STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vectors based on the PEG-mediated transformation method . The specific process of rice protoplast transformation can refer to the literature (Tang X, Zheng X, Qi YP, Zhang D, Cheng Y, Tang A, Voytas DF, Zhang Y.2016. Asingle transcript CRISPR-Cas9 system for efficient genome editing inplants. Molecular Plant , 9(7):1088-1091.) The experimental method disclosed in.

3. Detection of targeted editing of cytosine bases at specific sites in rice endogenous OsCDC48 and OsROC5 genes

After the rice protoplasts were transformed, they were cultured in the dark at 25°C for 48 hours, and the transformed cells were collected. The genomic DNA of the rice protoplasts was extracted by the CTAB method, and the DNA was used as a template for PCR amplification and Illumina high-throughput sequencing. For specific methods, refer to the literature (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 efficient genome editing and transcriptional repression in plants. Nature Plants, 3: 17018.).

Analysis of Illumina high-throughput sequencing results showed that 28.62% (OsCDC48-sgRNA01), 30.99% (OsROC5-gRNA04), 49.41% (OsROC5 -gRNA05) the editing efficiency of cytosine bases replaced by thymine bases (Figure 2: nCas9-PmCDA1). In particular, as a control group (according to reference reports (Komor AC, Kim YB, PackerMS, Zuris JA, Liu DR.2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature, 533 (7603 ): 420-424.), the rApobec1 cytosine deaminase is replaced by the PmCDA1 unit in the plant STU nCas9-PmCDA1 single transcription unit directed base editing backbone vector constructed by the present invention), and the cytosine editing efficiencies of the same target sequence are respectively 7.65% (OsCDC48-sgRNA01), 4.44% (OsROC5-gRNA04), 4.16% (OsROC5-gRNA05) (Figure 2: nCas9-rApobec1).

Based on the determined three cytosine base editing target sequences in rice, based on the Illumina high-throughput sequencing results, further analysis of the editing efficiency of replacing independent cytosine base sites with thymine bases at specific editing sites showed that: OsCDC48-sgRNA01 A total of 6 cytosine bases at C3, C4, C7, C8, C11, and C14 had effective thymine base substitution editing (Figure 3: nCas9-PmCDA1); a total of 2 cytosine bases at C2 and C5 at OsROC5-gRNA04 Effective thymine base substitution editing occurred at the base (Figure 4: nCas9-PmCDA1); a total of 3 cytosine bases C-1, C3, and C4 at OsROC5-gRNA05 had effective thymine base substitution editing (Figure 5: nCas9-PmCDA1). In particular, based on the plant STU nCas9-PmCDA1 single transcription unit directional base editing backbone carrier constructed by the present invention, at the C-1 site of -1bp at OsROC5-gRNA05, 37.94% of cytosine bases were replaced by The editing efficiency of thymine bases (Figure 5: nCas9-PmCDA1), a cytosine base editing event located outside the sgRNA target sequence interval, has not been achieved in existing studies. At the same time, as a control group, the editing window and editing efficiency of replacing independent cytosine base sites with thymine bases at the three tested rice cytosine base editing target sequences were significantly lower than those in the examples of the present invention (Figure 3 : nCas9-rApobec1; Figure 4: nCas9-rApobec1; Figure 5: nCas9-rApobec1;).

The above test results show that the plant STU nCas9-PmCDA1 single transcription unit directional base editing backbone vector constructed based on the present invention can effectively realize efficient directional editing of cytosine bases in specific regions of rice endogenous genes, and can further expand the scope of editing window.

Example 3 Based on the STU nCas9-PmCDA1 system, the creation and efficiency analysis of rice endogenous gene cytosine-directed base editing regenerated plants

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

The STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, and STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vectors successfully constructed in Example 2 and detected in rice protoplasts for directional modification activity were passed The competent cells of Agrobacterium EHA105 were transformed by the 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°C 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-edited recombinant expression vector

The recombinant expression vectors of STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, and STUnCas9-PmCDA1-OsROC5-gRNA05 were transformed into rice callus by Agrobacterium-mediated transformation. References for the specific process of transformation (Tang X, Zheng X, Qi YP, Zhang D, Cheng Y, TangA, Voytas DF, Zhang Y.2016.A single transcript CRISPR-Cas9 system for efficient genome editing in plants. Molecular Plant, 9( 7): 1088-1091.) The experimental method disclosed in.

3. Targeted base editing detection and efficiency analysis of rice endogenous gene STU nCas9-PmCDA1-sgRNA base editing recombinant expression vector stably transformed and regenerated plants

After the transformation, the resistant callus was induced into rice seedlings, and the genomic DNA of the transformed and regenerated rice seedlings was extracted, and the DNA was used as a template for PCR amplification and Sanger sequencing analysis. STU nCas9-PmCDA1-OsCDC48-sgRNA01, STU nCas9-PmCDA1-OsROC5-gRNA04, STU nCas9-PmCDA1-OsROC5-gRNA05 recombinant expression vectors The analysis of rice transformed and regenerated plants showed that three cytosine bases targeting rice OsCDC48 and OsROC5 endogenous genes Base editing target sequences achieved base editing efficiencies of 44.44% (OsCDC48-sgRNA01: 8/18), 100.00% (OsROC5-gRNA04: 26/26), and 68.75% (OsROC5-gRNA05: 11/16) (Figure 6 : nCas9-PmCDA1). The test results further illustrate that the plant STU nCas9-PmCDA1 single transcription unit directional base editing backbone carrier constructed by the present invention can effectively realize efficient directional editing of cytosine bases in specific regions of rice endogenous genes, and obtain base editing regenerated plants, It is a molecular tool to effectively realize the targeted editing of plant genome bases, which can improve the efficiency of genome engineering crop improvement.

Nucleotide and Amino Acid Sequences

Seq ID No.1: Nucleotide sequence of pZmUbi1-nCas9 ORF-PmCDA1-Poly A-sgRNA cloning scaffold-HSP T

CGCGCCTGCAGTGCAGCGTGACCCGGTCGTGCCCCTCTCTTGAGATAATGAGCATTGCATGTCTAAGTTATAAAAAATTACCACATATTTTTTTTGTCACACTTGTTTGAAGTGCAGTTTATCTATCTTTATACATATATTTAAACTTTACTCTACGAATAATATAATCTATAGTACTACAATAATATCAGTGTTTTAGAGAATCATATAAATGAACAGTTAGACATGGTCTAAAGGACAATTGAGTATTTTGACAACAGGACTCTACAGTTTTATCTTTTTAGTGTGCATGTGTTCTCCTTTTTTTTTGCAAATAGCTTCACCTATATAATACTTCATCCATTTTATTAGTACATCCATTTAGGGTTTAGGGTTAATGGTTTTTATAGACTAATTTTTTTAGTACATCTATTTTATTCTATTTTAGCCTCTAAATTAAGAAAACTAAAACTCTATTTTAGTTTTTTTATTTAATAATTTAGATATAAAATAGAATAAAATAAAGTGACTAAAAATTAAACAAATACCCTTTAAGAAATTAAAAAAACTAAGGAAACATTTTTCTTGTTTCGAGTAGATAATGCCAGCCTGTTAAACGCCGTCGACGAGTCTAACGGACACCAACCAGCGAACCAGCAGCGTCGCGTCGGGCCAAGCGAAGCAGACGGCACGGCATCTCTGTCGCTGCCTCTGGACCCCTCTCGAGAGTTCCGCTCCACCGTTGGACTTGCTCCGCTGTCGGCATCCAGAAATTGCGTGGCGGAGCGGCAGACGTGAGCCGGCACGGCAGGCGGCCTCCTCCTCCTCTCACGGCACCGGCAGCTACGGGGGATTCCTTTCCCACCGCTCCTTCGCTTTCCCTTCCTCGCCCGCCGTAATAAATAGACACCCCCTCCACACCCTCTTTCCCCAACCTCGTGTTGTTCGGAGCGCACACACACACAACCAGAACTCCCCCAAATCCACCCGTCGGCACCTCCGCTTCAAGGTACGCCGCTCG TCCTCCCCCCCCCCCCCTCTCTACCTTCTCAAGATCGGCGTTCCGGTCCATGGTTAGGGCCCGGTAGTTCTACTTCTGTTCATGTTTGTGTTAGATCCGTGTTTGTGTTAGATCCGTGCTACTAGCGTTCGTACACGGATGCGACCTGTACGTCAGACACGTTCTGATTGCTAACTTGCCAGTGTTTCTCTTTGGGGAATCCTGGGATGGCTCTAGCCGTTCCGCAGACGGGATCGATTTCATGATTTTTTTTGTTTCGTTGCATAGGGTTTGGTTTGCCCTTTTCCTTTATTTCAATATATGCCGTGCACTTGTTTGTCGGGTCATCTTTTCATGCTTTTTTTTGTCTTGGTTGTGATGATGTGGTCTGGTTGGGCGGTCGTTCAAGATCGGAGTAGAATTAATTCTGTTTCAAACTACCTGGTGGATTTATTAATTTTGGATCTGTATGTGTGTGCCATACATATTCATAGTTACGAATTGAAGATGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGCGGGTTTTACTGATGCATATACAGAGATGCTTTTTGTTCGCTTGGTTGTGATGATGTGGTGTGGTTGGGCGGTCGTTCATTCGTTCAAGATCGGAGTAGAATACTGTTTCAAACTACCTGGTGTATTTATTAATTTTGGAACTGTATGTGTGTGTCATACATCTTCATAGTTACGAGTTTAAGATGGATGGAAATATCGATCTAGGATAGGTATACATGTTGATGTGGGTTTTACTGATGCATATACATGATGGCATATGCAGCATCTATTCATATGCTCTAACCTTGAGTACCTATCTATTATAATAAACAAGTATGTTTTATAATTATTTTGATCTTGATATACTTGGATGATGGCATATGCAGCAGCTATATGTGGATTTTTTTAGCCCTGCCTTCATACGCTATTTATTTGCTTGGTACTGTTTCTTTTGTCGATGCTCACCCTGTTGTTTGGTGTTACTTCTGC AGCCTGCAGGATGGATAAGAAGTACTCTATCGGACTCGCTATCGGAACTAACTCTGTGGGATGGGCTGTGATCACCGATGAGTACAAGGTGCCATCTAAGAAGTTCAAGGTTCTCGGAAACACCGATAGGCACTCTATCAAGAAAAACCTTATCGGTGCTCTCCTCTTCGATTCTGGTGAAACTGCTGAGGCTACCAGACTCAAGAGAACCGCTAGAAGAAGGTACACCAGAAGAAAGAACAGGATCTGCTACCTCCAAGAGATCTTCTCTAACGAGATGGCTAAAGTGGATGATTCATTCTTCCACAGGCTCGAAGAGTCATTCCTCGTGGAAGAAGATAAGAAGCACGAGAGGCACCCTATCTTCGGAAACATCGTTGATGAGGTGGCATACCACGAGAAGTACCCTACTATCTACCACCTCAGAAAGAAGCTCGTTGATTCTACTGATAAGGCTGATCTCAGGCTCATCTACCTCGCTCTCGCTCACATGATCAAGTTCAGAGGACACTTCCTCATCGAGGGTGATCTCAACCCTGATAACTCTGATGTGGATAAGTTGTTCATCCAGCTCGTGCAGACCTACAACCAGCTTTTCGAAGAGAACCCTATCAACGCTTCAGGTGTGGATGCTAAGGCTATCCTCTCTGCTAGGCTCTCTAAGTCAAGAAGGCTTGAGAACCTCATTGCTCAGCTCCCTGGTGAGAAGAAGAACGGACTTTTCGGAAACTTGATCGCTCTCTCTCTCGGACTCACCCCTAACTTCAAGTCTAACTTCGATCTCGCTGAGGATGCAAAGCTCCAGCTCTCAAAGGATACCTACGATGATGATCTCGATAACCTCCTCGCTCAGATCGGAGATCAGTACGCTGATTTGTTCCTCGCTGCTAAGAACCTCTCTGATGCTATCCTCCTCAGTGATATCCTCAGAGTGAACACCGAGATCACCAAGGCTCCACTCTCAGCTTCTATGATCAAGAGATACGATGAGCACCACCAG GATCTCACACTTCTCAAGGCTCTTGTTAGACAGCAGCTCCCAGAGAAGTACAAAGAGATTTTCTTCGATCAGTCTAAGAACGGATACGCTGGTTACATCGATGGTGGTGCATCTCAAGAAGAGTTCTACAAGTTCATCAAGCCTATCCTCGAGAAGATGGATGGAACCGAGGAACTCCTCGTGAAGCTCAATAGAGAGGATCTTCTCAGAAAGCAGAGGACCTTCGATAACGGATCTATCCCTCATCAGATCCACCTCGGAGAGTTGCACGCTATCCTTAGAAGGCAAGAGGATTTCTACCCATTCCTCAAGGATAACAGGGAAAAGATTGAGAAGATTCTCACCTTCAGAATCCCTTACTACGTGGGACCTCTCGCTAGAGGAAACTCAAGATTCGCTTGGATGACCAGAAAGTCTGAGGAAACCATCACCCCTTGGAACTTCGAAGAGGTGGTGGATAAGGGTGCTAGTGCTCAGTCTTTCATCGAGAGGATGACCAACTTCGATAAGAACCTTCCAAACGAGAAGGTGCTCCCTAAGCACTCTTTGCTCTACGAGTACTTCACCGTGTACAACGAGTTGACCAAGGTTAAGTACGTGACCGAGGGAATGAGGAAGCCTGCTTTTTTGTCAGGTGAGCAAAAGAAGGCTATCGTTGATCTCTTGTTCAAGACCAACAGAAAGGTGACCGTGAAGCAGCTCAAAGAGGATTACTTCAAGAAAATCGAGTGCTTCGATTCAGTTGAGATTTCTGGTGTTGAGGATAGGTTCAACGCATCTCTCGGAACCTACCACGATCTCCTCAAGATCATTAAGGATAAGGATTTCTTGGATAACGAGGAAAACGAGGATATCTTGGAGGATATCGTTCTTACCCTCACCCTCTTTGAAGATAGAGAGATGATTGAAGAAAGGCTCAAGACCTACGCTCATCTCTTCGATGATAAGGTGATGAAGCAGTTGAAGAGAAGAAGATACACTGGTTGGGGAAGGCTCTCAA GAAAGCTCATTAACGGAATCAGGGATAAGCAGTCTGGAAAGACAATCCTTGATTTCCTCAAGTCTGATGGATTCGCTAACAGAAACTTCATGCAGCTCATCCACGATGATTCTCTCACCTTTAAAGAGGATATCCAGAAGGCTCAGGTTTCAGGACAGGGTGATAGTCTCCATGAGCATATCGCTAACCTCGCTGGATCTCCTGCAATCAAGAAGGGAATCCTCCAGACTGTGAAGGTTGTGGATGAGTTGGTGAAGGTGATGGGAAGGCATAAGCCTGAGAACATCGTGATCGAAATGGCTAGAGAGAACCAGACCACTCAGAAGGGACAGAAGAACTCTAGGGAAAGGATGAAGAGGATCGAGGAAGGTATCAAAGAGCTTGGATCTCAGATCCTCAAAGAGCACCCTGTTGAGAACACTCAGCTCCAGAATGAGAAGCTCTACCTCTACTACCTCCAGAACGGAAGGGATATGTATGTGGATCAAGAGTTGGATATCAACAGGCTCTCTGATTACGATGTTGATCATATCGTGCCACAGTCATTCTTGAAGGATGATTCTATCGATAACAAGGTGCTCACCAGGTCTGATAAGAACAGGGGTAAGAGTGATAACGTGCCAAGTGAAGAGGTTGTGAAGAAAATGAAGAACTATTGGAGGCAGCTCCTCAACGCTAAGCTCATCACTCAGAGAAAGTTCGATAACTTGACTAAGGCTGAGAGGGGAGGACTCTCTGAATTGGATAAGGCAGGATTCATCAAGAGGCAGCTTGTGGAAACCAGGCAGATCACTAAGCACGTTGCACAGATCCTCGATTCTAGGATGAACACCAAGTACGATGAGAACGATAAGTTGATCAGGGAAGTGAAGGTTATCACCCTCAAGTCAAAGCTCGTGTCTGATTTCAGAAAGGATTTCCAATTCTACAAGGTGAGGGAAATCAACAACTACCACCACGCTCACGATGCTTACCTTAACGCTGTTGTTGGAACCGCTCT CATCAAGAAGTATCCTAAGCTCGAGTCAGAGTTCGTGTACGGTGATTACAAGGTGTACGATGTGAGGAAGATGATCGCTAAGTCTGAGCAAGAGATCGGAAAGGCTACCGCTAAGTATTTCTTCTACTCTAACATCATGAATTTCTTCAAGACCGAGATTACCCTCGCTAACGGTGAGATCAGAAAGAGGCCACTCATCGAGACAAACGGTGAAACAGGTGAGATCGTGTGGGATAAGGGAAGGGATTTCGCTACCGTTAGAAAGGTGCTCTCTATGCCACAGGTGAACATCGTTAAGAAAACCGAGGTGCAGACCGGTGGATTCTCTAAAGAGTCTATCCTCCCTAAGAGGAACTCTGATAAGCTCATTGCTAGGAAGAAGGATTGGGACCCTAAGAAATACGGTGGTTTCGATTCTCCTACCGTGGCTTACTCTGTTCTCGTTGTGGCTAAGGTTGAGAAGGGAAAGAGTAAGAAGCTCAAGTCTGTTAAGGAACTTCTCGGAATCACTATCATGGAAAGGTCATCTTTCGAGAAGAACCCAATCGATTTCCTCGAGGCTAAGGGATACAAAGAGGTTAAGAAGGATCTCATCATCAAGCTCCCAAAGTACTCACTCTTCGAACTCGAGAACGGTAGAAAGAGGATGCTCGCTTCTGCTGGTGAGCTTCAAAAGGGAAACGAGCTTGCTCTCCCATCTAAGTACGTTAACTTTCTTTACCTCGCTTCTCACTACGAGAAGTTGAAGGGATCTCCAGAAGATAACGAGCAGAAGCAACTTTTCGTTGAGCAGCACAAGCACTACTTGGATGAGATCATCGAGCAGATCTCTGAGTTCTCTAAAAGGGTGATCCTCGCTGATGCAAACCTCGATAAGGTGTTGTCTGCTTACAACAAGCACAGAGATAAGCCTATCAGGGAACAGGCAGAGAACATCATCCATCTCTTCACCCTTACCAACCTCGGTGCTCCTGCTGCTTTCAAGTACTTCGATACAACC ATCGATAGGAAGAGATACACCTCTACCAAAGAAGTGCTCGATGCTACCCTCATCCATCAGTCTATCACTGGACTCTACGAGACTAGGATCGATCTCTCACAGCTCGGTGGTGATTCAAGGGCTGATCCTAAGAAGAAGAGGAAGGTTGGAGACGACGGAGGTGGCGGTACAGGAGGGGGTGGGTCCGCTGAGTATGTCAGGGCGTTGTTCGACTTCAATGGAAACGACGAGGAAGATCTGCCTTTTAAAAAGGGAGATATTCTCAGGATCAGAGATAAGCCGGAAGAACAATGGTGGAACGCTGAAGACTCTGAAGGTAAGAGAGGTATGATTCTTGTCCCCTACGTCGAGAAGTATTCGGGTGACTATAAAGACCACGATGGAGATTATAAGGACCACGATATAGATTATAAGGATGATGATGATAAGAGCGGAATGACCGATGCAGAGTACGTCAGGATTCATGAGAAACTTGACATCTACACGTTTAAGAAACAGTTTTTCAACAACAAAAAATCTGTTAGTCACCGCTGTTACGTGCTGTTCGAATTGAAACGCAGAGGTGAGAGGAGAGCCTGCTTTTGGGGCTATGCCGTCAACAAGCCGCAAAGCGGCACAGAAAGGGGCATTCACGCGGAGATATTTAGCATTAGAAAGGTCGAGGAATACCTTCGGGATAATCCCGGGCAATTCACTATCAATTGGTACTCTTCATGGTCCCCGTGTGCAGATTGCGCTGAAAAGATACTGGAGTGGTATAATCAAGAACTCAGAGGAAACGGTCACACCCTCAAGATTTGGGCTTGCAAGCTTTACTACGAGAAAAATGCAAGGAACCAGATCGGCCTCTGGAACTTGCGCGACAACGGCGTGGGGTTGAATGTGATGGTGTCGGAGCATTACCAGTGCTGCCGGAAGATATTCATTCAGTCGTCACATAATCAATTGAACGAGAATAGGTGGCTCGAAAAAACCCTGAAGCGGGCCGAGAAGTGGAGGA GTGAACTCTCGATAATGATCCAGGTTAAAATACTGCATACTACCAAATCTCCGGCGGTGGGACCGAAGAAGAAGCGCAAGGTGGGGACCATGACTAATCTCTCAGATATAATCGAGAAGGAAACAGGAAAGCAACTGGTCATCCAAGAATCGATTTTGATGCTTCCCGAAGAAGTCGAAGAAGTTATAGGAAATAAGCCCGAGTCTGACATACTGGTTCACACAGCGTACGATGAAAGTACGGACGAGAATGTCATGTTGCTGACATCGGACGCACCTGAATACAAGCCTTGGGCTCTGGTCATACAAGATAGTAACGGAGAAAATAAGATTAAAATGCTTTCAGGTGGCTCCCCAAAGAAGAAACGCAAGGTTTGAGGATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGGAGACCTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGAAACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAATAAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCTTCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACT GATAGCTGTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACATATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTGGTCTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCAACAAAGCACCAGTGGTCTAGTGGTAGAATAGTACCCTGCCACGGTACAGACCCGGGTTCGATTCCCGGCTGGTGCAGGATCCATATGAAGATGAAGATGAAATATTTGGTGTGTCAAATAAAAAGCTTGTGTGCTTAAGTTTGTGTTTTTTTCTTGGCTTGTTGTGTTATGAATTTGTGGCTTTTTCTAATATTAAATGAATGTAAGATCACATTATAATGAATAAACAAATGTTTCTATAATCCATTGTGAATGTTTTGTTGGATCTCTTCTGCAGCATATAACTACTGTATGTGCTATGGTATGGACTATGGAATATGATTAAAGATAAG

Seq ID No.2: Amino acid sequence of nCas9-PmCDA1 nuclease-cytidine deaminase fusion protein expression cassette

MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSRADPKKKRKVGDDGGGGTGGGGSAEYVRALFDFNGNDEEDLPFKKGDILRIRDKPEEQWWNAEDSEGKRGMILVPYVEKYSGDYKDHDGDYKDHDIDYKDDDDKSGMTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKWRSELSIMIQVKILHTTKSPAVGPKKKRKVGTMTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSPKKKRKV。

sequence listing

<110> University of Electronic Science and Technology

<120> A plant genome-directed base editing backbone vector and its application

<130> 20180001

<141> 2018-11-23

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8556

<212>DNA

<213> 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

atccaccccgt cggcacctcc gcttcaaggt acgccgctcg tcctcccccc ccccccctct 1020

ctaccttctc aagatcggcg ttccggtcca tggttagggc ccggtagttc tacttctgtt 1080

catgtttgtg ttagatccgt gtttgtgtgtta gatccgtgct actagcgttc gtacacggat 1140

gcgacctgta cgtcagacac gttctgattg ctaacttgcc agtgtttctc tttggggaat 1200

cctgggatgg ctctagccgt tccgcagacg ggatcgattt catgattttt tttgtttcgt 1260

tgcataggt 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 agagggggagg 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 tattagcat 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

tctaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaagca 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 ggccagggggg 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 aagtttgtgtgtttttttcttg 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

<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 Lys Arg Lys Val

1780 1785

<210> 3

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 3

cgacatccgc aagtaccagg 20

<210> 4

<211> 22

<212>DNA

<213> Artificial Sequence

<400> 4

agtacactgt ttccccgtat gt 22

<210> 5

<211> 19

<212>DNA

<213> Artificial Sequence

<400> 5

gctgctggtg agtgctgat 19

<210> 6

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 6

acccattggg agtgtcttgc 20

<210> 7

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 7

gaccagccag cgtctggcgc 20

<210> 8

<211> 20

<212>DNA

<213> Artificial Sequence

<400> 8

gcagctggct gagggtgcat 20

<210> 9

<211> 19

<212>DNA

<213> Artificial Sequence

<400> 9

agccagctgc ttacaaaac 19

<210> 10

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 10

tgcagaccag ccagcgtctg gcgc 24

<210> 12

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 12

aaacgcgcca gacgctggct ggtc 24

<210> 13

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 13

tgcagcagct ggctgagggt gcat 24

<210> 13

<211> 24

<212>DNA

<213> Artificial Sequence

<400> 13

aaacatgcac cctcagccag ctgc 24

<210> 14

<211> 23

<212>DNA

<213> Artificial Sequence

<400> 14

tgcaagccag ctgcttacaa aac 23

<210> 15

<211> 23

<212>DNA

<213> 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.