CN113717960A - Novel Cas9 protein, CRISPR-Cas9 genome directed editing vector and genome editing method - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a novel Cas9 protein, a CRISPR-Cas9 genome directional editing vector and a genome editing method. The technical problem to be solved by the invention is that fewer CRISPR-Cas systems have been developed at present. The technical scheme for solving the technical problems is to provide a novel Cas9 nuclease protein which has the capacity of recognizing a 5 '-NGAAA-3' PAM locus. Can simply, quickly and efficiently carry out directional modification on the genome of a plant, even realize the editing, directional modification and expression regulation of the genome aiming at various positions (particularly non-coding sequences) in the genome, and has better application prospect.

Description

Novel Cas9 protein, CRISPR-Cas9 genome directed editing vector and genome editing method

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to a novel Cas9 protein which can identify a 5 '-NGAAA-3' PAM locus, a CRISPR-Cas9 genome directional editing vector and a genome editing method.

Background introduction

The functional identification and breeding application of the genes related to the specific traits of the plants depend on the acquisition of reliable mutant germplasm materials to a certain extent, and the existing mutant germplasm materials are mainly obtained based on the collection of natural mutants and a physical and chemical factor mutagenesis strategy. Meanwhile, gene mutation caused by the creation of the mutant is random and is difficult to direct mutation aiming at specific genes, which is a huge brake for biological breeding, gene function research and species de novo domestication. With the development of molecular manipulation technology, genome editing technology represented by ZFN, TALEN and CRISPR-Cas has been effectively applied to mutant germplasm material innovation. The CRISPR-Cas system is characterized by simple structure, high editing efficiency and strong expansibility, and is a genome editing technology which is most widely applied in the future.

The most commonly used Cas9 system is mainly the SpCas9 system derived from Streptococcus pyogenes (Streptococcus pyogenes), which mainly recognizes the PAM site as 5 '-NGG-3'. The SpCas9 system has limited editing on the rich A/T region, has fewer selectable sites and greatly influences the practical application. Compared with a huge CRISPR-Cas member library, the CRISPR-Cas system developed at present still appears to be more limited, and new members with excellent editing performance still need to be mined. Therefore, the search of a new Cas system and the application of the new Cas system in genome editing have very important significance and are also important strategic requirements for the industrialized development of the biotechnology in China. The search for a new CRISPR-Cas system with high specificity, high editing activity, undifferentiated PAM recognition, which is naturally evolved, in an abundant prokaryotic genome library, and the possibility of developing and exploring it from scratch as a genome editing tool is a work that is currently urgently needed in the art but is full of challenges.

Disclosure of Invention

The technical problem to be solved by the invention is that fewer CRISPR-Cas systems have been developed at present. The technical means for solving the technical problems is to provide a novel Cas9 nuclease protein. The Cas9 protein is:

(a) the amino acid sequence is shown as SeqID No. 1; or:

(b) the amino acid sequence has one or more amino acid substitutions, deletions or additions compared with the sequence shown in SeqID No.1, and has the same or similar biological functions.

Further, the Cas9 protein described above having the same or similar biological function means having at least one of the following activities:

an activity of binding to a guide RNA, an activity of binding to a specific site of a target sequence under the guidance of a guide RNA, an endonuclease activity, an activity of binding to a specific site of a target sequence and cleaving a nucleic acid under the guidance of a guide RNA, or a PAM site recognition; further, the PAM site is characterized by any of 5 '-NGAAA-3', N representation A, G, C, T.

Further, the amino acid sequence of the Cas9 protein is obtained by performing D13A mutation and/or H858A mutation on SEQ ID No. 1.

The invention also provides a coding gene of the Cas9 protein.

Further, the nucleotide sequence of the encoding gene of the Cas9 protein is shown as Seq ID No. 2.

The D13A mutation changed the nucleotide at this position from GAT to GCT and the H858A mutation changed the nucleotide at this position from CAT to GCT.

The invention also provides an expression vector containing the coding gene.

Wherein, the expression vector contains a Cas9 protein expression unit and has the structure of ZmUbi1-LacCas 9-NLS-AtHSP.

Wherein, the ZmUbi1 is a maize Ubi1 promoter. The nucleotide sequence of the promoter of Ubi1 in maize is shown in Seq ID No.3, 1-1996. The NLS is a nuclear localization sequence. The coding nucleotide sequence of NLS is shown in 6110-6208 in Seq ID No. 3. The AtHSP is an Arabidopsis HSP terminator. The AtHSP coding nucleotide sequence is shown in 6218-6467 in Seq ID No. 3.

Further, the nucleotide sequence of the expression unit of the Cas9 protein in the expression vector is shown as 1-6467 in Seq ID No. 3.

The expression vector also comprises a sgRNA clone and a transcription unit, and can co-express Cas9 protein and sgRNA.

Further, the structure of the sgRNA clone and transcription unit is OsU6-ccdB-sgRNA scafffold-TTTTTT.

Wherein OsU6 is rice OsU6 promoter, and the nucleotide sequence is represented by 6477-6722 in Seq ID No. 3. The sgRNA scaffold is a sgRNA framework, and the sequence can be shown as 7367-7505 in the Seq ID No.3, as well as the sequence ID No.4 or as shown as the Seq ID No. 5; the ccdB is an Escherichia coli lethal gene, and the coding nucleotide sequence is shown as 6729-7353 in Seq ID No. 3. The TTTTTT is a sgRNA transcription termination signal, and the nucleotide sequence is shown as 7506-7511 in Seq ID No. 3.

Furthermore, the nucleotide sequence of the sgRNA scaffold module of the sgRNA cloning and transcription unit in the expression vector is 6477-7511 in Seq ID No.3, 4 or 5

Wherein, the expression vector also contains a hygromycin resistance screening gene.

The invention also provides application of the expression vector in constructing a CRISPR-Cas9 gene editing system.

The invention also provides a CRISPR-Cas9 gene editing method. The method uses the expression vector to provide the activity of the Cas9 protein for the CRISPR-Cas9 gene editing system.

Further, the method comprises the following steps:

a. constructing a framework vector: constructing the expression vector of any one of claims 6 to 11 as a CRISPR-Cas9 backbone vector;

b. constructing a directional gene editing vector: designing a gene editing site and sgRNA aiming at a target gene to be edited, synthesizing a primer pair of the sgRNA, annealing the primer pair to form double-stranded DNA with a sticky end, and replacing a ccdB element in a CRISPR-Cas9 skeleton vector with the double-stranded DNA to obtain a directional editing expression vector;

c. targeted gene editing:

b, transforming the cell to be edited by using the directional editing expression vector obtained in the step b to obtain a cell or a plant with directional gene editing;

or;

b, transforming the protoplast by using the directional editing expression vector obtained in the step b, detecting the gene directional editing condition of the protoplast, and culturing the protoplast successfully subjected to directional editing to obtain a directional gene editing plant;

or;

and c, transferring the directional editing expression vector obtained in the step b into Agrobacterium tumefaciens (Agrobacterium tumefaciens), and introducing the vector into a target plant by an Agrobacterium-mediated genetic transformation method to obtain a directional gene editing plant.

Further, step b of the above method may be performed by the following specific steps:

a) the target DNA region of the plant genome to be edited is determined, a PAM site characteristic region which can be identified by the Cas9 nuclease protein is analyzed, and a 20bpDNA sequence adjacent to the 3' end of the PAM structure is selected as a specific modification target sequence; the PAM site feature 5 '-NGAAA-3', N represents any one of A, G, C, T;

b) synthesizing a forward oligonucleotide chain with the characteristics of 5 '-GTGTG-NX-3' and a reverse oligonucleotide chain with the characteristics of 5 '-AGAC-NX-3' according to the selected specific modified target sequence, wherein N represents any one of A, G, C, T, X is an integer, and when the first position of the selected target sequence is G, X is 19; when the first position of the selected target sequence is not G, X ═ 20, where NX in the forward oligonucleotide strand and NX in the reverse oligonucleotide have reverse complementary characteristics; obtaining a complementary oligonucleotide double-stranded fragment by annealing;

c) and replacing the ccdB element in the CRISPR-Cas9 skeleton vector with the double-stranded DNA to obtain the directional editing expression vector.

The invention has the beneficial effects that: the invention obtains a novel Cas9 protein, and the Cas9 protein has the capacity of recognizing a 5 '-NGAAA-3' PAM locus. Through the codon optimization of the Cas12a protein gene, the protein coding gene suitable for plant CRISPR-Cas9 gene editing is obtained. On the basis, a novel efficient CRISPR-Cas9 plant genome directed modification skeleton vector is also provided, a diversified toolkit is developed, and the plant genome directed modification can be simply, quickly and efficiently carried out. Genome editing, targeted modification and expression regulation can even be achieved for diverse locations in the genome (particularly non-coding sequences). Experiments show that the CRISPR-Cas9 skeleton vector is a system with a novel PAM site efficient editing capacity and has a good application prospect.

Drawings

Fig. 1, new CRISPR-Cas9 systematic mining construction scheme. A. New CRISPR-Cas9 system mining flow chart; B. structural schematic diagram of different gRNAs of LacCas9 system

Fig. 2, results of detection of different gRNA cleavage activity of LacCas9 system based on transient protoplast transformation. A. RFLP analysis results comparing gRNA V1.0 and gRNA V2.0 editing activities; B. high throughput sequencing analysis results comparing gRNA V2.0 and gRNA V3.0 editing activities.

FIG. 3, results of rice endogenous gene editing based on the LacCas9 system transformed transiently by protoplast.

FIG. 4, editing specificity analysis of LacCas9 system in rice.

FIG. 5, comparison of the editing activity of LacCas9 system with SpCas9-NG, SpRY and LbCas12a systems. A. LacCas9, SpCas9-NG, SpRY and LbCas12a system edit positions; b C, LacCas9 system and LbCas12a system editing activity comparison; d E, LacCas9 system compared with SpCas9-NG, SpRY system editing activity.

FIG. 6, analysis of editing activity of LacCas9 system in rice T0 generation plants. A. Statistics of editing efficiency of the LacCas9 system in rice T0 generation plants; B. and (3) analyzing mutation conditions of rice T0 generation mutants created by a LacCas9 system.

Fig. 7, analysis of editing activity of LacCas9 system in wheat.

Fig. 8, LacCas9 cytosine single base editing efficiency analysis. A. A LacCas9 cytosine single base editor schematic; B. analyzing the editing efficiency of a LacCas9 cytosine single-base editing system in rice protoplasts; C. and (3) analyzing the editing efficiency of the LacCas9 cytosine single-base editing system in wheat protoplasts.

Fig. 9, analysis of adenine single base editing efficiency by LacCas 9. A. LacCas9 adenine single base editor diagram; B. LacCas9 adenine single base machine editing efficiency in rice protoplasts was analyzed.

FIG. 10, analysis of the editing efficiency of LacCas9 single base editor in rice T0 generation plants. A. Analyzing the editing efficiency of a LacCas9 cytosine single-base editor in rice T0 generation plants; B. analyzing mutation conditions of LacCas9 cytosine single base editing plants; C. analyzing the editing efficiency of a LacCas9 adenine single-base editor in rice T0 generation plants; D. and (3) analyzing mutation conditions of plants edited by adenine single base of LacCas 9.

Fig. 11, LacCas9 gene regulation system example. A. A schematic diagram of a LacCas9 gene activation regulation system; B. a LacCas9 gene activation regulation system regulation result; C. schematic diagram of LacCas9 gene suppression regulation system; D. the LacCas9 gene suppression regulation system regulates the result.

The specific implementation mode is as follows:

the potential for discovery is enormous, since CRISPR-Cas systems are relatively limited as members of the development and successful application of systems that are ubiquitous in (archaic) bacteria. In order to develop a CRISPR-Cas new system, the invention adopts a strategy of crossing bioinformatics and experimental science. The method comprises the steps of firstly excavating possible CRISPR-Cas gene loci by a bioinformatics method, analyzing Cas proteins in the CRISPR-Cas gene loci, analyzing tracrRNA and crRNA to obtain sequence information of the CRISPR-Cas gene loci, then carrying out BLAST analysis on a spacer sequence to obtain PAM information of a new member, and finally constructing and verifying a system by experiments.

On the basis of a data mining strategy, bacterial and archaeal genome databases are effectively compared, and a new CRISPR-Cas member with genome editing capacity is screened, a new efficient plant CRISPR-Cas genome editing system is constructed by taking rice as a model, and feasible application of the system in rice functional genome analysis and germplasm innovation is realized.

Through the work, a new CRISPR-Cas9 member capable of identifying a 5 '-NGAAA-3' PAM site is obtained, and a new gene editing system with high specificity and high editing activity is constructed on the basis.

The amino acid sequence of the new Cas9 protein is shown as SeqID No.1 and is named as LacCas9 nuclease. Meanwhile, substitution, deletion or addition of one or several amino acids is performed on the basis of the amino acid sequence shown in SeqID No.1, and amino acids having the same or similar biological functions are also easily obtained by those skilled in the art.

The LacCas9 nuclease shown in the above Seq ID No.1 can be used for constructing a conventional LacCas9 genome editing system. In the example provided by the invention, a LacCas9 genome editing system for recognizing a 5 '-NGAAA-3' PAM locus is successfully constructed, and the targeted modification and editing of endogenous genes in rice are successfully realized. Further, the above-mentioned having the same or similar biological functions means having at least one of the following activities:

activity of binding to a guide RNA, activity of binding to a specific site on the target sequence under the guidance of a guide RNA, endonuclease activity, activity of binding to a specific site on the target sequence and cleaving nucleic acid under the guidance of a guide RNA, or recognition of a PAM site. The PAM site is characterized by any of 5 '-NGAAA-3', N stands for A, G, C, T.

For example, in the examples provided herein, two variants are disclosed in SEQ ID NO: 1, and performing amino acid conversion on the basis of the amino acid sequence shown in the figure to obtain the mutant protein with similar functions.

One is that D13A mutation is carried out on Seq ID No.1, the protein is different from LacCas9 nuclease in that only single-stranded nucleic acid can be cut, and a nick is formed on double-stranded nucleic acid, and the protein can be called as LacCas9 nickase in the invention. The mutant protein obtained by mutating aspartic acid Asp at the 13 th position into alanine Ala can be used for constructing a single base editing system. Of course, when a single-base editing system is constructed, it is generally used in combination with an active domain commonly used for single-base editing, such as cytosine deaminase.

The other is the D13A mutation and H858A in Seq ID No.1, which is distinguished from LacCas9 nuclease by loss of only the activity of cleaving nucleic acids. The mutant protein can be used for constructing a transcription regulation gene editing system. Of course, in the construction of transcription regulatory systems, it is generally used in combination with domains that are commonly used in the art for transcription regulation.

The LacCas9 genome editing system (including single base editing system or transcription regulating system) further provided by the invention contains a Cas9 protein expression unit, namely, the expression of the LacCas9 encoding gene is controlled by a promoter. Generally, the sequence encoding the nuclear localization sequence NLS is arranged after the coding gene of LacCas9 in the Cas9 protein expression unit. In one example of the present invention, a three-segment NLS structure (3 XNLS) is used, which is obtained by repeating the SRADPKKKRKV (Seq ID No.22) fragment three times. Wherein the PKKKRKV fragment plays a role in nuclear localization, and the SRAD fragment is an linker sequence. The typical structure of the Cas9 protein expression unit is promoter-LacCas 9-NLS-terminator.

The LacCas9 genome editing system (comprising a single-base editing system or a transcription regulating system) also comprises sgRNA cloning and a transcription unit, and is used for expressing sgRNA which can be matched with the LacCas9 protein for use. One of the core components of the sgRNA cloning and transcription unit is the sgRNA scaffold component. For a better implementation of the invention, the invention also constructed 3 sgRNA scaffold modules whose nucleotide sequences are indicated by 6477-7511, Seq ID No.4 or Seq ID No.5 in Seq ID No.3, respectively.

The present invention is further illustrated by the following examples, which are intended to illustrate the invention but not to limit it further, and should not be construed as limiting the scope of the invention.

Example 1 data mining and construction of LacCas9 System

1. Data mining of the new Cas9 system:

in earlier studies, data mining analysis was performed on the genome data of microorganisms derived from the genome database of the National Center for Biotechnology Information, NCBI (https:// www.ncbi.nlm.nih.gov). 287 known Cas proteins, 257, 745 putative Cas proteins and 3593 CRISPR loci are found in 33, 834 (archaic) bacterial genomes, and after tracrRNA and PAM sequence prediction is carried out on 328 of them which are predicted to be Type II CRISPR-Cas systems, 100 are preliminarily selected for subsequent experiments. 4 CRISPR loci with new PAM recognition potential are analyzed and tested subsequently, related vectors are constructed and editing activity evaluation is performed, and 1 new CRISPR-Cas9 system (fig. 1) which can recognize different PAMs and has higher activity is discovered. The system is derived from Lactobacillus (Lactobacillus spp., GenBank: AP 011548). Its CRISPR locus, comprising four Cas-associated protein genes, is analyzed, annotated in order as Cas9, Cas1, Cas2 and Csn2 genes. The Cas9 gene in the locus has the length of 4092bp, encodes 1363 amino acids, is named as LacCas9 protein, and has the amino acid sequence (Seq ID No. 1): MTKLNQPYGIGLDIGSNSIGFAVVDANSHLLRLKGETAIGARLFREGQSAADRRGSRTTRRRLSRTRWRLSFLRDFFAPHITKIDPDFFLRQKYSEISPKDKDRFKYEKRLFNDRTDAEFYEDYPSMYHLRLHLMTHTHKADPREIFLAIHHILKSRGHFLTPGAAKDFNTDKVDLEDIFPALTEAYAQVYPDLELTFDLAKADDFKAKLLDEQATPSDTQKALVNLLLSSDGEKEIVKKRKQVLTEFAKAITGLKTKFNLALGTEVDEADASNWQFSMGQLDDKWSNIETSMTDQGTEIFEQIQELYRARLLNGIVPAGMSLSQAKVADYGQHKEDLELFKTYLKKLNDHELAKTIRGLYDRYINGDDAKPFLREDFVKALTKEVTAHPNEVSEQLLNRMGQANFMLKQRTKANGAIPIQLQQRELDQIIANQSKYYDWLAAPNPVEAHRWKMPYQLDELLNFHIPYYVGPLITPKQQAESGENVFAWMVRKDPSGNITPYNFDEKVDREASANTFIQRMKTTDTYLIGEDVLPKQSLLYQKYEVLNELNNVRINNECLGTDQKQRLIREVFERHSSVTIKQVADNLVAHGDFARRPEIRGLADEKRFLSSLSTYHQLKEILHEAIDDPTKLLDIENIITWSTVFEDHTIFETKLAEIEWLDPKKINELSGIRYRGWGQFSRKLLDGLKLGNGHTVIQELMLSNHNLMQILADETLKETMTELNQDKLKTDDIEDVINDAYTSPSNKKALRQVLRVVEDIKHAANGQDPSWLFIETADGTGTAGKRTQSRQKQIQTVYANAAQELIDSAVRGELEDKIADKASFTDRLVLYFMQGGRDIYTGAPLNIDQLSHYDIDHILPQSLIKDDSLDNRVLVNATINREKNNVFASTLFAGKMKATWRKWHEAGLISGRKLRNLMLRPDEIDKFAKGFVARQLVETRQIIKLTEQIAAAQYPNTKIIAVKAGLSHQLREELDFPKNRDVNHYHHAFDAFLAARIGTYLLKRYPKLAPFFTYGEFAKVDVKKFREFNFIGALTHAKKNIIAKDTGEIVWDKERDIRELDRIYNFKRMLITHEVYFETADLFKQTIYAAKDSKERGGSKQLIPKKQGYPTQVYGGYTQESGSYNALVRVAEADTTAYQVIKISAQNASKIASANLKSREKGKQLLNEIVVKQLAKRRKNWKPSANSFKIVIPRFGMGTLFQNAKYGLFMVNSDTYYRNYQELWLSRENQKLLKKLFSIKYEKTQMNHDALQVYKAIIDQVEKFFKLYDINQFRAKLSDAIERFEKLPINTDGNKIGKTETLRQILIGLQANGTRSNVKNLGIKTDLGLLQVGSGIKLDKDTQIVYQSPSGLFKRRIPLADL

By analyzing the DNA sequence at the 3' end of the locus, we have derived the crRNA sequence of the system: the sequence is 36bp long, and the direction is consistent with the direction of a Cas9 gene; by comparing the crRNA sequence with the locus sequence, promoter analysis was performed on the locus, predicting its tracrRNA sequence: the tracrRNA sequence is located between the Cas9 gene and the Cas1 gene, is 138bp long, and has the sequence direction opposite to that of the Cas9 gene. The Spacer sequence was compared to the phage genome and the PAM recognized by this system was presumed to be 5 '-NGAAA-3'. The tracrRNA and crRNA can be ligated into a guide RNA by ligation with an RNA linker. Thus, a new Cas9 gene editing system with 5 '-NGAAA-3' as identified PAM can be obtained, and the system is named as LacCas9 system.

2. LacCas9 genome editing system construction:

according to the Cas9 gene sequence information obtained by data mining, the rice codon preference optimization is carried out on the gene, and a Kingsry company is entrusted to synthesize a LacCas9 gene fragment. Three guide rnas (sgrna scaffold) were designed by analyzing tracrRNA and crRNA, and named gRNA V1.0, gRNA V2.0, and gRNA V3.0, respectively. The three guide RNAs differ by the pairing length difference between tracrRNA and crRNA (fig. 1). The DNA sequences of the three guide RNAs were synthesized by Kinsley. The three guide RNA sequences are shown (7367-7505, Seq ID No.4 or Seq ID No.5 in Seq ID No. 3).

The skeleton carrier of the system is constructed in two steps.

Step 1: a pZHHY 988 vector (related information and sequence of the vector are shown in http:// www.addgene.org/search/advanced/. Mixing pZHY98The vector 8 was digested with the FD-SdaI and FD-HindIII restriction enzymes as follows: 10 XFastduest Green Buffer, 3. mu.L; FD-SdaI, 1 μ L; FD-HindIII, 1 μ L; pZHY988 plasmid DNA (2. mu.g), 1. mu.L; ddH₂O, 24 μ L; 1-2 h at 37 ℃. The size of the digested fragment is about 4205bp and 11647bp, and a 11647bp fragment is recovered by using an AxyPrep DNA gel recovery kit.

The product of pZHHY 988 enzyme digestion recovery and the LacCas9 gene fragment are assembled by a Gibson assembly method, and the reaction system is as follows: gibson Assembly Mix, 15 μ L; pZHY988 fragment, 2. mu.L (50 ng); LacCas9 gene fragment, 3 μ L (10 times molar amount of vector); 50 ℃ for 1 h. After the reaction is finished, 6 mu L of Gibson assembly product is taken to transform escherichia coli strain DB3.1 competent cells, an LB solid plate containing Kan (50mg/L) antibiotics is coated, and the mixture is cultured for 18-22h at 37 ℃.

And (3) selecting a monoclonal colony on the plate to dilute and mix uniformly in 50 mu L of sterilized deionized water, taking 5 mu L of the bacterial liquid as a template, and taking LacCas9-Seq5 (5'-GGCGAACGGCACAAGGTCCA-3' Seq ID No.6) and ZY065-RB (5'-TTCTAATAAACGCTCTTTTCTCT-3' Seq ID No.7) as upstream and downstream primers to carry out positive identification on colony PCR, wherein the length of a positive amplification product is about 1600 bp. The sequence of the obtained vector is further verified through enzyme digestion and sequencing, so that CRISPR-Cas9-LacCas9-step1 is constructed.

Step 2: taking a CRISPR-Cas9-LacCas9-step1 vector as a framework, and carrying out enzyme digestion by using FD-SacI and FD-HindIII restriction enzymes, wherein the enzyme digestion system is as follows: 10 XFastduest Green Buffer, 3. mu.L; FD-SacI, 1 μ L; FD-HindIII, 1 μ L; CRISPR-Cas9-LacCas9-step1 plasmid DNA (2 μ g), 1 μ L; ddH₂O, 24 μ L; 1-2 h at 37 ℃. The size of the enzyme cutting fragment is about 1231bp and 14675bp, and a 11647bp fragment is recovered by using an AxyPrep DNA gel recovery kit.

The product of pZHHY 988 enzyme digestion recovery and the LacCas9 gene fragment are assembled by a Gibson assembly method, and the reaction system is as follows: gibson Assembly Mix, 15 μ L; CRISPR-Cas9-LacCas9-step1 enzyme cutting fragment, 2 μ L (50 ng); 3 μ L of guide RNA gene fragment (10 times molar amount of vector); 50 ℃ for 1 h. After the reaction is finished, 6 mu L of Gibson assembly product is taken to transform escherichia coli strain DB3.1 competent cells, an LB solid plate containing Kan (50mg/L) antibiotics is coated, and the mixture is cultured for 18-22h at 37 ℃.

And (3) selecting a monoclonal colony on the plate to dilute and mix uniformly in 50 mu L of sterilized deionized water, taking 5 mu L of the bacterial liquid as a template, taking gRNA-Seq (5'-GGTACCGGTCTCAGTCTCAGGTAGATGTCAGATCA-3' Seq ID No.8) and ZY065-RB (5'-TTCTAATAAACGCTCTTTTCTCT-3' Seq ID No.12) as upstream and downstream primers, and carrying out positive identification on colony PCR, wherein the lengths of positive amplification products are about 240bp, 270bp and 284 bp. The sequence of the obtained vector is further verified through enzyme digestion and sequencing, so that the vectors of CRISPR-Cas9-LacCas9-gRV01, CRISPR-Cas9-LacCas9-gRV02 and CRISPR-Cas9-LacCas9-gRV03 are constructed. Wherein the nucleotide sequence of CRISPR-Cas9-LacCas9-gRV02 is shown as Seq ID No.3, wherein 1-1996 is a maize ZmUbi1 promoter; 2021-6109 is the LacCas9 nuclease nucleotide sequence; 6110-6208 is the 3' NLS coding sequence; 6218-6467 is an Arabidopsis HSP terminator; 6477-6722 is the OsU6 promoter; 6729-7353 is the ccdB gene; 7367 SgRNA scaffold (gRNA V2.0) at 7505; 7506-7511 is the sgRNA transcription termination signal. The CRISPR-Cas9-LacCas9-gRV01 and CRISPR-Cas9-LacCas9-gRV03 replace sgRNA scaffold (gRNA V2.0) in CRISPR-Cas9-LacCas9-gRV02 with sgRNA scaffold (gRNA V1.0, nucleotide sequence is shown in Seq ID No. 4) and sgRNA scaffold (gRNA V3.0, nucleotide sequence is shown in Seq ID No.5)

3.CRISPR-Cas 9-LacCas9 guide RNA assay

Design of Gene editing site AGAAA-sgRNA01 for Rice endogenous albino Gene OsPDS (

Underlined PstI cleavage site, bolded PAM site), TGAAA-sgRNA02(

Thickening to PAM locus), plant type gene OsDEP1 design locus CGAAA-sgRNA01(

Bold to PAM site), artificially synthesized primer AGAAA-sgRNA01-F (5'-GTGTGAGTCCTGGCAAACAACCTGC-3', Seq ID N)o.12) and AGAAA-sgRNA01-R (5'-AGACGCAGGTTGTTTGCCAGGACTC-3', Seq ID No. 13); TGAAA-sgRNA02-F (5'-GTGTGTGGCATTTCTACCTTATCGA-3', Seq ID No.14) and TGAAA-sgRNA02-R (5'-AGACTCGATAAGGTAGAAATGCCAC-3', Seq ID No. 15); CGAAA-sgRNA01-F (5'-GTGTGTCCCGAGCGCGGAGTACGTA-3', Seq ID No.16) and CGAAA-sgRNA01-R (5'-AGACTACGTACTCCGCGCTCGGGAC-3', Seq ID No.17), each of which was tested for guide RNA editing activity. 20 mu L of each primer pair is uniformly mixed, denatured at 95 ℃ for 10min, and naturally cooled and annealed to form target gene double-stranded DNA with a sticky end. The annealing fragments and CRISPR-Cas9-LacCas9-gRV01, CRISPR-Cas9-LacCas9-gRV02 and CRISPR-Cas9-LacCas9-gRV03 vectors are cloned and connected by a Golden Gate method, wherein the Golden Gate reaction system is as follows: 10 XT 4 ligase buffer, 2. mu.L; bsa I, 1 μ L; t4 DNA ligase, 1. mu.L; backbone vector (100ng), 1 μ L; annealed product (10mM), 2. mu.L; ddH₂O, 13. mu.L. The reaction procedure was as follows: (37 ℃, 5 min; 16 ℃, 10 min). times.10 cycles; at 37 ℃ for 10 min; 80 ℃ for 10 min. After the program is finished, 6 mu of LGolden Gate reaction product is added into escherichia coli DH5a competent cells for transformation, and is plated on a plate containing Kan (50mg/L) LB solid medium for 18-22 h. Identifying positive colonies by monoclonal colony PCR (the system is as described above), and verifying the positive colonies by extracting plasmids, enzyme digestion, sequencing and the like to obtain AGAAA-sgRNA01 directional editing expression vectors including CRISPR-Cas9-LacCas9-gRV01-OsPDS-sgR01 and CRISPR-Cas9-LacCas9-gRV02-OsPDS-sgR 01; TGAAA-sgRNA02 directionally edits expression vectors including CRISPR-Cas9-LacCas9-gRV02-OsPDS-sgR02 and CRISPR-Cas9-LacCas9-gRV03-OsPDS-sgR 02; CGAAA-sgRNA01 directionally edits expression vectors including CRISPR-Cas9-LacCas9-gRV02-OsDEP1-sgR01 and CRISPR-Cas9-LacCas9-gRV03-OsDEP1-sgR 01.

Through a PEG-mediated rice protoplast preparation and transformation system (Zhang et al, A high grade effective rice plasmid green tissue plasmid expression and stuck light/chloroplatst-related processes plant Methods,2011,7(1):30), 30 mu g CRISPR-Cas9-LacCas9-gRV01-OsPDS-sgR01, CRISPR-Cas 9-Lac695Cas 2-gRV 02-OsPDS-sgR01, CRISPR-Cas9-LacCas9-gRV02-OsPDS-sgR02, CRISPR-Cas9-LacCas 9-9-OsPDS-9, CRISPR-9-LacCas 9-9-OsPDS 9, CRISPR-9-LacCas 9-OsgRNA 9, and the target gene AGRNA-9, the target gene DNA is extracted and the target gene is subjected to targeted PCR, the target gene is extracted and the target gene is amplified by the PEG-Cas DNA-9, the CRISPR-9-OsAAG DNA, the target gene is extracted and the target gene is subjected to the target gene, the amplification primers were as follows: AGAAA-sgRNA01 site (OsPDS-F: 5'-TTCGCAAGTAGCAGCATCCA-3', Seq ID No.18)), OsPDS-R: 5'-TGGTTTAGTGGGTTGTTCACTG-3', Seq ID No.19)), the amplification system was as follows: 10 XTaq Buffer, 2.5. mu.L; taq DNA Polymerase (2.5U/. mu.L), 0.4. mu.L; dNTPs (10mM), 0.5. mu.L; OsPDS-F, 0.5 μ L; OsPDS-R, 0.5 μ L; gDNA (100 ng/. mu.L), 2. mu.L; ddH2O, 18.6. mu.L. The amplification procedure was as follows: at 95 ℃ for 3 min; (95 ℃, 30 s; 56 ℃,20 s; 72 ℃, 40 s). times.28 cycles; 72 ℃ for 5 min; 12 ℃ for 5 min. The obtained PCR amplification product is about 550bp, the PCR product is cut by PstI enzyme, if the PCR product is a wild type, two bands of about 190bp and 109bp can be obtained, and if the PCR product is a mutant, only one resistant band of 299bp can be obtained. The cleavage activity of the CRISPR-Cas9-LacCas9-gRV01 and CRISPR-Cas9-LacCas9-gRV02 editing systems can be preliminarily evaluated by detecting the amounts of the resistance bands and the enzyme cutting bands. After the CRISPR-Cas9-LacCas9-gRV01 is transformed by protoplast, no resistance band is detected by PCR \ RFLP analysis, which indicates that the editing activity of the system is low; and after the CRISPR-Cas9-LacCas9-gRV02 system is detected, an obvious resistance zone is observed, and the system is proved to be capable of realizing effective genome editing in rice protoplasts. Further testing the CRISPR-Cas9-LacCas9-gRV02 and CRISPR-Cas9-LacCas9-gRV03 editing systems, and finding that the two systems can realize effective genome editing through protoplast transformation and high-throughput sequencing analysis, wherein the editing efficiency of the CRISPR-Cas9-LacCas9-gRV02 system is 21.6% -24.4%; the editing efficiency of the CRISPR-Cas9-LacCas9-gRV03 is 21.6-26.9% (FIG. 2). Because the editing efficiency of the two systems is basically consistent, the CRISPR-Cas9-LacCas9-gRV02 system is selected for subsequent experiments.

Example 2 plant endogenous Gene Targeted modification based on the LacCas9 System

1. Endogenous gene targeted modification and editing characteristic analysis of LacCas9 system in rice

Further, another 13 sites were selected in rice, for a total of 16 5 '-NGAAA-3' PAM sites, for genome editing activity validation based on the LacCas9 system. The editing efficiency of a total of 14 PAM sites by protoplast transformation and high throughput sequencing analysis is shown in figure 3.

The specificity was verified by designing mismatches at positions 1-20 of the spacer and detecting the editing activity of the LacCas9 system at the endogenous site in rice. Through protoplast transformation and high throughput sequencing analysis, the editing activity was greatly reduced in all 1bp mismatch spaders as shown in FIG. 4. Specifically, the mismatches at positions 2, 6, 13, 15, 18 and 19 of the spacer basically lose the editing activity of the LacCas9 system, while the mismatches at the other positions still enable the LacCas9 system to generate certain editing activity, but the highest editing activity is about 30% of the original editing activity. The above results show that the LacCas9 system has better specificity, and the editing activity is obviously reduced under the condition of 1bp mismatch in the spacer, which is better than that of the SpCas9 system.

Since PAM recognized by LacCas9 system overlaps with Cas12a system (recognizing 5 '-TTTC-3' PAM), SpCas9-NG system (recognizing 5 '-NGA-3' PAM) and SpRY system (recognizing 5 '-NGA-3' PAM), we compared the editing efficiency of LacCas9 system and the three systems in rice. Through transient protoplast transformation and high throughput sequencing analysis, we found that the LacCas9 system had editing efficiency consistent with or better than that of the LbCas12a system at the selected four sites. The editing efficiency (19.4-57.3%) of the LacCas9 system at the S4, S10 and S16 sites is higher than that of the LbCas12a system (1.2-33.7%), and the difference is obvious; at the site of S2, the two systems have substantially identical editing efficiency (35.8% for LacCas9 system editing efficiency and 33.7% for LbCas12a system editing efficiency). Combining the editing efficiency of all sites, the editing efficiency of the LacCas9 system is higher than that of the LbCas12a system (FIG. 5).

The CRISPR-Cas9-LacCas9-gRV02-OsPDS-sgR01, CRISPR-Cas9-LacCas9-gRV02-OsPDS-sgR02, CRISPR-Cas9-LacCas9-gRV02-OsDEP1-sgR01 and the vector are transferred into Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 by a freeze-thaw method, and positive clones are screened. The above-described vector was introduced into Nipponbare rice by an Agrobacterium-mediated genetic transformation method to obtain a stable transgenic plant (Hiei et al, Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the rooting of the T-DNA.1994.plant Journal,6(2): 271-282.). The obtained transgenic lines were tested one by one for directional mutants using the same test method in protoplasts as described above, and the Sanger test results showed (fig. 6): the mutation efficiency of CRISPR-Cas9-LacCas9-gRV02-OsPDS-sgR01 is 44.0%, the mutation efficiency of CRISPR-Cas9-LacCas9-gRV02-OsPDS-sgR02 is 68.0%, and the mutation efficiency of CRISPR-Cas9-LacCas9-gRV02-OsDEP1-sgR01 is 45.0%. The results show that the CRISPR-Cas9-LacCas9-gRV02 editing system can realize effective genome editing.

Plants with OsPDS-sgRNA02 site oriented editing were randomly picked for sequencing, and genotype analysis showed that (FIG. 6): the shearing mode of the CRISPR-Cas9-LacCas9-gRV02 editing system is basically consistent with that of the SpCas9 system, namely, 1bp insertion or deletion is generated at 3 and 4 bases which are mostly adjacent to the PAM.

2. Endogenous gene-directed modification of the LacCas9 system in wheat

The LacCas9 system is tested in wheat, and the OsU6 promoter in the CRISPR-Cas9-LacCas9-gRV02 system is replaced by TaU6 promoter to construct the wheat CRISPR-Cas9-LacCas9-gRV02 system. TaU6 (sequence reference https:// www.addgene.org/search/catalog/plasmids/.

Taking a CRISPR-Cas9-LacCas9-gRV02 vector as a framework, and carrying out enzyme digestion by using FD-SacI and FD-BamHI restriction enzymes, wherein the enzyme digestion system is as follows: 10 XFastduest Green Buffer, 3. mu.L; FD-SacI, 1 μ L; FD-BamHI, 1. mu.L; CRISPR-Cas9-LacCas9-gRV02 plasmid DNA (2 μ g), 1 μ L; ddH₂O, 24 μ L; 1-2 h at 37 ℃. The size of the enzyme digestion fragment is about 481bp and 15491bp, and the about 15491bp fragment is recovered by using an AxyPrep DNA gel recovery kit.

The product of enzyme digestion recovery of CRISPR-Cas9-LacCas9-gRV02 and TaU6 gene fragment are assembled by a Gibson assembly method, and the reaction system is as follows: gibson Assembly Mix, 15 μ L; CRISPR-Cas9-LacCas9-gRV02 enzyme-cleaved fragment, 2 μ L (50 ng); TaU6 gene fragment, 3 μ L (10 times molar weight of vector); 50 ℃ for 1 h. After the reaction is finished, 6 mu L of Gibson assembly product is taken to transform escherichia coli strain DB3.1 competent cells, an LB solid plate containing Kan (50mg/L) antibiotics is coated, and the mixture is cultured for 18-22h at 37 ℃.

And (3) selecting a monoclonal colony on the plate to dilute and mix uniformly in 50 mu L of sterilized deionized water, taking 5 mu L of the bacterial liquid as a template, taking gRNA-Seq (5'-GGTACCGGTCTCAGTCTCAGGTAGATGTCAGATCA-3', Seq ID No.8) and ZY065-RB (5'-TTCTAATAAACGCTCTTTTCTCT-3', Seq ID No.7) as upstream and downstream primers, and carrying out positive identification on colony PCR, wherein the length of a positive amplification product is about 270 bp. And further carrying out enzyme digestion and sequencing to verify the sequence of the obtained vector, thereby constructing and obtaining the CRISPR-Cas9-LacCas9-Ta-gRV 02.

Sites are selected for expression vector construction and protoplast transformation, and through high-throughput analysis, the editing efficiency of the LacCas9 system in wheat is 1.2-10.0%. The above results demonstrate that the LacCas9 system can achieve efficient genome editing in wheat (fig. 7).

Example 3 Single base editing tool development based on LacCas9 System

1. Cytosine deaminase is used to achieve cytosine → thymine transversion:

according to the cDNA sequence of PmCDA1-UGI gene published in Nishida et al, 2016 (Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A. targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. science.2016Sep 16; 353(6305): aaf8729.) LacCas9(D13A) -PmA 1-UGI gene was synthesized by rice codon optimization. The enzyme digestion is carried out by using FD-SdaI and FD-BspTI restriction enzymes by using a CRISPR-Cas9-LacCas9-gRV02 vector and a CRISPR-Cas9-LacCas9-Ta-gRV02 as frameworks, wherein the enzyme digestion system is as follows: 10 XFastduest Green Buffer, 3. mu.L; FD-SdaI, 1 μ L; FD-BspTI, 1 μ L; backbone vector plasmid DNA (2. mu.g), 1. mu.L; ddH₂O, 24 μ L; 1-2 h at 37 ℃. The sizes of the enzyme digestion fragments are about 4267bp, 11705bp, 4267bp and 11823bp, and the fragments of about 11705bp and 11823bp are recovered by an AxyPrep DNA gel recovery kit.

The enzyme-cleaved and recovered products of CRISPR-Cas9-LacCas9-gRV02 and CRISPR-Cas9-LacCas9-Ta-gRV02 are assembled with LacCas9(D13A) -PmCDA1-UGI by a Gibson assembly method, and the reaction system is as follows: gibson Assembly Mix, 15 μ L; backbone vector restriction, 2. mu.L (50 ng); LacCas9(D13A) -PmCDA1-UGI gene fragment, 3 μ L (molar amount is 10 times of the vector); 50 ℃ for 1 h. After the reaction is finished, 6 mu L of Gibson assembly product is taken to transform escherichia coli strain DB3.1 competent cells, an LB solid plate containing Kan (50mg/L) antibiotics is coated, and the mixture is cultured for 18-22h at 37 ℃.

And (3) selecting a monoclonal colony on the plate to dilute and mix uniformly in 50 mu L of sterilized deionized water, taking 5 mu L of the bacterial liquid as a template, taking gRNA-Seq (5'-GGTACCGGTCTCAGTCTCAGGTAGATGTCAGATCA-3', Seq ID No.8) and ZY065-RB (5'-TTCTAATAAACGCTCTTTTCTCT-3', Seq ID No.7) as upstream and downstream primers, and carrying out positive identification on colony PCR, wherein the length of a positive amplification product is about 270 bp. And further carrying out enzyme digestion and sequencing verification on the sequence of the obtained vector, thereby constructing LacCas9-OsCBE and LacCas9-TaCBE skeleton vectors.

Selecting sites in endogenous genes of rice and wheat to construct expression vectors. The LacCas9-OsCBE system has 1.4% -32.4% C to T editing activity at the eight selected sites in rice by protoplast transformation and high throughput sequencing analysis. In wheat, the C to T base editing activity of the LacCas9-TaCBE system at the four sites selected was 4.2% to 20.2% (fig. 8).

2. Adenine deaminase was applied to achieve adenine → guanine transversion:

EcTadA 7.10-LacCas9(D13A) and ABE8E-Lac 13A 25 (D13 Cas 38) genes were synthesized by rice codon optimization according to the cDNA sequences of the EcTadA 7.10 and ABE8E genes published by Richter et al, Nature Biotechnology journal (Richter MF, Zhao KT, Eton E, Lapinaite A, Newby GA, Thuronyi BW, Wilson C, Koblan LW, Zeng J, Bauer DE, Doudna JA, Liu DR.phase-analyzed evolution of an adenosine base with improved Cas domain compatibility and activity.Nature Biotechnology.2020J. mu.L; 38(7):883 and 891.) in 2020 et al. Taking a CRISPR-Cas9-LacCas9-gRV02 vector as a framework, and carrying out enzyme digestion by using FD-SdaI and FD-BspTI restriction endonucleases, wherein the enzyme digestion system is as follows: 10 × Fastdiget Green Buffer, 3. mu.L; FD-SdaI, 1 μ L; FD-BspTI, 1 μ L; CRISPR-Cas9-LacCas9-gRV02 plasmid DNA (2 μ g), 1 μ L; ddH₂O, 24 μ L; 1-2 h at 37 ℃. The size of the enzyme digestion fragment is about 4267bp and 11705bp, and a 11705bp fragment is recovered by using an AxyPrep DNA gel recovery kit.

The product of enzyme digestion and recovery of CRISPR-Cas9-LacCas9-gRV02, the gene ecTadA 7.10-LacCas9(D13A) and the gene ABE8e-LacCas9(D13A) are assembled by a Gibson assembly method, and the reaction system is as follows: gibson Assembly Mix, 15 μ L; backbone vector restriction, 2. mu.L (50 ng); ecTadA 7.10 (or ABE8e) gene fragment, 3. mu.L (molar amount 10 times of vector); 50 ℃ for 1 h. After the reaction is finished, 6 mu L of Gibson assembly product is taken to transform escherichia coli strain DB3.1 competent cells, an LB solid plate containing Kan (50mg/L) antibiotics is coated, and the mixture is cultured for 18-22h at 37 ℃.

And (3) selecting a monoclonal colony on the plate to dilute and mix uniformly in 50 mu L of sterilized deionized water, taking 5 mu L of the bacterial liquid as a template, and taking Cas9-Seq1 (5'-TGATGGCATATGCAGCAGCT-3', Seq ID No.20) and LacCas9-Seq1(5'-TGTGTGTCGGACGGGGTGGC-3', Seq ID No.21) as upstream and downstream primers to carry out positive identification on colony PCR, wherein the length of a positive amplification product is about 1400 bp. The sequences of the obtained vectors are further verified by enzyme digestion and sequencing, so that LacCas9-ABE-V1.0 and LacCas9-ABE-V2.0 framework vectors are constructed (FIG. 9).

Selecting sites in the rice endogenous gene and constructing an expression vector. The A to G editing activity of the LacCas9-ABE-V1.0 and LacCas9-ABE-V2.0 systems at selected three sites was 0.0% to 0.4% in rice by protoplast transformation and high throughput sequencing analysis (FIG. 9).

The editing efficiency of the LacCas9 system in rice T0 generation was analyzed based on stable transformation of rice. As shown in FIG. 10, the editing efficiency of the LacCas9-OsCBE system in rice T0 individual plant is 19.1% -38.0%; the editing efficiency of the LacCas9-ABE-V2.0 system in rice T0 individual plants is 14.3%.

Example 4 Gene regulatory tool development based on LacCas9 System

According to the SRDX gene sequence published in 2012 by Mahfouz et al (Mahfouz MM, Li L, Piatek M, Fang X, Mansour H)Bangarusamy DK, Zhu JK.Targeted translational repression using a polymeric TALE-SRDX repression or protein.plant Mol biol.2012Feb; 78(3):311-21.), the KRAB gene sequence published by Urrutia et al in 2003 (Urrutia R.KRAB-stabilizing zinc-finger repressors proteins. genome biol.2003; 4(10):231.) and the two genes are constructed into a chimeric gene KRAB-SRDX, and a mutant protein LacCas9(D13A, H858A) -KRAB-SRDX fusion gene sequence of LacCas9 is synthesized through rice codon optimization. A mutant protein LacCas9(D13A, H858A) -TV fusion gene sequence of LacCas9 was synthesized from the TV gene sequences published in Li et al 2017 (Li Z, Zhang D, Xiong X, Yan B, Xie W, Sheen J, Li JF. A potential Cas9-derived gene activator for plant and mammalian cells. Nat plants.2017 Dec; 3(12): 930) 936.). Based on a CRISPR-Cas9-LacCas9-gRV02 vector, the enzyme digestion is carried out by using FD-SdaI and FD-BspTI restriction enzymes, and the enzyme digestion system is as follows: 10 XFastduest Green Buffer, 3. mu.L; FD-SdaI, 1 μ L; FD-BspTI, 1 μ L; backbone vector plasmid DNA (2. mu.g), 1. mu.L; ddH₂O, 24 μ L; 1-2 h at 37 ℃. The size of the enzyme digestion fragment is about 4267bp and 11705bp, and a 11705bp fragment is recovered by using an AxyPrep DNA gel recovery kit.

The product of the CRISPR-Cas9-LacCas9-gRV02 enzyme digestion recovery is assembled with LacCas9(D13A, H858A) -KRAB-SRDX and LacCas9(D13A, H858A) -TV gene sequences by a Gibson assembly method, and the reaction system is as follows: gibson Assembly Mix, 15 μ L; backbone vector restriction, 2. mu.L (50 ng); gene fragment, 3 μ L (10 times molar amount of vector); 50 ℃ for 1 h. After the reaction is finished, 6 mu L of Gibson assembly product is taken to transform escherichia coli strain DB3.1 competent cells, an LB solid plate containing Kan (50mg/L) antibiotics is coated, and the mixture is cultured for 18-22h at 37 ℃.

And (3) selecting a monoclonal colony on the plate to dilute and mix uniformly in 50 mu L of sterilized deionized water, taking 5 mu L of the bacterial liquid as a template, taking gRNA-Seq (5'-GGTACCGGTCTCAGTCTCAGGTAGATGTCAGATCA-3', Seq ID No.8) and ZY065-RB (5'-TTCTAATAAACGCTCTTTTCTCT-3', Seq ID No.7) as upstream and downstream primers, and carrying out positive identification on colony PCR, wherein the length of a positive amplification product is about 270 bp. The sequences of the obtained vectors were further verified by enzyme digestion and sequencing, and LacCas9-KS and LacCas9-TV backbone vectors were constructed (FIG. 11).

The activity of the dLacCas9-KS system was verified by OsALT2, OsGW7 and OsWx genes in rice. Through protoplast transient transformation and real-time quantitative PCR analysis, obvious reduction of expression level (the expression level is 1.8-28.7% of that of the control) is generated at all the selected five sites. OsALT2, OsNRT1.1 and OsmiR528 genes in rice are selected to carry out directional activation test on the dLacCas9-TV system. Through protoplast transient transformation and real-time quantitative PCR analysis, we find that the dLacCas9-TV system leads to the increase of the expression level of the corresponding gene in the selected four sites. Compared with the control, the gene expression level after the treatment of the system is increased to 1.7-3.0 times of the control (FIG. 11). The above results demonstrate that the LacCas9 system can achieve precise gene expression regulation.

Sequence listing

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Met Thr Lys Leu Asn Gln Pro Tyr Gly Ile Gly Leu Asp Ile Gly Ser

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

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

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Ser Ala Ala Asp Arg Arg Gly Ser Arg Thr Thr Arg Arg Arg Leu Ser

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Arg Thr Arg Trp Arg Leu Ser Phe Leu Arg Asp Phe Phe Ala Pro His

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Asn Asp Arg Thr Asp Ala Glu Phe Tyr Glu Asp Tyr Pro Ser Met Tyr

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

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Glu Asp Ile Phe Pro Ala Leu Thr Glu Ala Tyr Ala Gln Val Tyr Pro

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Asp Leu Glu Leu Thr Phe Asp Leu Ala Lys Ala Asp Asp Phe Lys Ala

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

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Val Asn Leu Leu Leu Ser Ser Asp Gly Glu Lys Glu Ile Val Lys Lys

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

245 250 255

Thr Lys Phe Asn Leu Ala Leu Gly Thr Glu Val Asp Glu Ala Asp Ala

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Ser Asn Trp Gln Phe Ser Met Gly Gln Leu Asp Asp Lys Trp Ser Asn

275 280 285

Ile Glu Thr Ser Met Thr Asp Gln Gly Thr Glu Ile Phe Glu Gln Ile

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Gln Glu Leu Tyr Arg Ala Arg Leu Leu Asn Gly Ile Val Pro Ala Gly

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Met Ser Leu Ser Gln Ala Lys Val Ala Asp Tyr Gly Gln His Lys Glu

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Asp Leu Glu Leu Phe Lys Thr Tyr Leu Lys Lys Leu Asn Asp His Glu

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Leu Ala Lys Thr Ile Arg Gly Leu Tyr Asp Arg Tyr Ile Asn Gly Asp

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Asp Ala Lys Pro Phe Leu Arg Glu Asp Phe Val Lys Ala Leu Thr Lys

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Glu Val Thr Ala His Pro Asn Glu Val Ser Glu Gln Leu Leu Asn Arg

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Met Gly Gln Ala Asn Phe Met Leu Lys Gln Arg Thr Lys Ala Asn Gly

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Ala Ile Pro Ile Gln Leu Gln Gln Arg Glu Leu Asp Gln Ile Ile Ala

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Asn Gln Ser Lys Tyr Tyr Asp Trp Leu Ala Ala Pro Asn Pro Val Glu

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Ala His Arg Trp Lys Met Pro Tyr Gln Leu Asp Glu Leu Leu Asn Phe

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His Ile Pro Tyr Tyr Val Gly Pro Leu Ile Thr Pro Lys Gln Gln Ala

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Glu Ser Gly Glu Asn Val Phe Ala Trp Met Val Arg Lys Asp Pro Ser

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Gly Asn Ile Thr Pro Tyr Asn Phe Asp Glu Lys Val Asp Arg Glu Ala

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

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

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Glu Val Leu Asn Glu Leu Asn Asn Val Arg Ile Asn Asn Glu Cys Leu

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Gly Thr Asp Gln Lys Gln Arg Leu Ile Arg Glu Val Phe Glu Arg His

565 570 575

Ser Ser Val Thr Ile Lys Gln Val Ala Asp Asn Leu Val Ala His Gly

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

595 600 605

Phe Leu Ser Ser Leu Ser Thr Tyr His Gln Leu Lys Glu Ile Leu His

610 615 620

Glu Ala Ile Asp Asp Pro Thr Lys Leu Leu Asp Ile Glu Asn Ile Ile

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Thr Trp Ser Thr Val Phe Glu Asp His Thr Ile Phe Glu Thr Lys Leu

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Ala Glu Ile Glu Trp Leu Asp Pro Lys Lys Ile Asn Glu Leu Ser Gly

660 665 670

Ile Arg Tyr Arg Gly Trp Gly Gln Phe Ser Arg Lys Leu Leu Asp Gly

675 680 685

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

690 695 700

Asn His Asn Leu Met Gln Ile Leu Ala Asp Glu Thr Leu Lys Glu Thr

705 710 715 720

Met Thr Glu Leu Asn Gln Asp Lys Leu Lys Thr Asp Asp Ile Glu Asp

725 730 735

Val Ile Asn Asp Ala Tyr Thr Ser Pro Ser Asn Lys Lys Ala Leu Arg

740 745 750

Gln Val Leu Arg Val Val Glu Asp Ile Lys His Ala Ala Asn Gly Gln

755 760 765

Asp Pro Ser Trp Leu Phe Ile Glu Thr Ala Asp Gly Thr Gly Thr Ala

770 775 780

Gly Lys Arg Thr Gln Ser Arg Gln Lys Gln Ile Gln Thr Val Tyr Ala

785 790 795 800

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

805 810 815

Asp Lys Ile Ala Asp Lys Ala Ser Phe Thr Asp Arg Leu Val Leu Tyr

820 825 830

Phe Met Gln Gly Gly Arg Asp Ile Tyr Thr Gly Ala Pro Leu Asn Ile

835 840 845

Asp Gln Leu Ser His Tyr Asp Ile Asp His Ile Leu Pro Gln Ser Leu

850 855 860

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

865 870 875 880

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

885 890 895

Met Lys Ala Thr Trp Arg Lys Trp His Glu Ala Gly Leu Ile Ser Gly

900 905 910

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

915 920 925

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

930 935 940

Lys Leu Thr Glu Gln Ile Ala Ala Ala Gln Tyr Pro Asn Thr Lys Ile

945 950 955 960

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

965 970 975

Phe Pro Lys Asn Arg Asp Val Asn His Tyr His His Ala Phe Asp Ala

980 985 990

Phe Leu Ala Ala Arg Ile Gly Thr Tyr Leu Leu Lys Arg Tyr Pro Lys

995 1000 1005

Leu Ala Pro Phe Phe Thr Tyr Gly Glu Phe Ala Lys Val Asp Val Lys

1010 1015 1020

Lys Phe Arg Glu Phe Asn Phe Ile Gly Ala Leu Thr His Ala Lys Lys

1025 1030 1035 1040

Asn Ile Ile Ala Lys Asp Thr Gly Glu Ile Val Trp Asp Lys Glu Arg

1045 1050 1055

Asp Ile Arg Glu Leu Asp Arg Ile Tyr Asn Phe Lys Arg Met Leu Ile

1060 1065 1070

Thr His Glu Val Tyr Phe Glu Thr Ala Asp Leu Phe Lys Gln Thr Ile

1075 1080 1085

Tyr Ala Ala Lys Asp Ser Lys Glu Arg Gly Gly Ser Lys Gln Leu Ile

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Pro Lys Lys Gln Gly Tyr Pro Thr Gln Val Tyr Gly Gly Tyr Thr Gln

1105 1110 1115 1120

Glu Ser Gly Ser Tyr Asn Ala Leu Val Arg Val Ala Glu Ala Asp Thr

1125 1130 1135

Thr Ala Tyr Gln Val Ile Lys Ile Ser Ala Gln Asn Ala Ser Lys Ile

1140 1145 1150

Ala Ser Ala Asn Leu Lys Ser Arg Glu Lys Gly Lys Gln Leu Leu Asn

1155 1160 1165

Glu Ile Val Val Lys Gln Leu Ala Lys Arg Arg Lys Asn Trp Lys Pro

1170 1175 1180

Ser Ala Asn Ser Phe Lys Ile Val Ile Pro Arg Phe Gly Met Gly Thr

1185 1190 1195 1200

Leu Phe Gln Asn Ala Lys Tyr Gly Leu Phe Met Val Asn Ser Asp Thr

1205 1210 1215

Tyr Tyr Arg Asn Tyr Gln Glu Leu Trp Leu Ser Arg Glu Asn Gln Lys

1220 1225 1230

Leu Leu Lys Lys Leu Phe Ser Ile Lys Tyr Glu Lys Thr Gln Met Asn

1235 1240 1245

His Asp Ala Leu Gln Val Tyr Lys Ala Ile Ile Asp Gln Val Glu Lys

1250 1255 1260

Phe Phe Lys Leu Tyr Asp Ile Asn Gln Phe Arg Ala Lys Leu Ser Asp

1265 1270 1275 1280

Ala Ile Glu Arg Phe Glu Lys Leu Pro Ile Asn Thr Asp Gly Asn Lys

1285 1290 1295

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

1300 1305 1310

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

1315 1320 1325

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

1330 1335 1340

Ile Val Tyr Gln Ser Pro Ser Gly Leu Phe Lys Arg Arg Ile Pro Leu

1345 1350 1355 1360

Ala Asp Leu

<210> 2

<211> 4089

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgacaaagc tgaaccagcc atacggcatc ggcctcgata ttggctcaaa ctctatcggc 60

ttcgcggtgg tcgacgccaa ttcccacctc ctgcgcctga agggcgagac agccatcggc 120

gccaggctct tccgcgaggg acagtcagcc gcggatcgga ggggctctag gaccacacgg 180

aggcgcctct cccggacaag gtggcgcctc agcttcctgc gcgacttctt cgcgccacat 240

atcaccaaga ttgaccctga tttcttcctc cgccagaagt actccgagat cagccctaag 300

gacaaggatc ggttcaagta cgagaagagg ctgttcaacg accgcaccga tgccgagttc 360

tacgaggatt acccatcaat gtaccacctg aggctccatc tgatgaccca cacacataag 420

gccgacccga gggagatttt cctcgccatc caccatattc tcaagtctcg gggccacttc 480

ctcacacctg gagcggccaa ggatttcaat accgacaagg tggatctgga ggacatcttc 540

ccagccctca cagaggcgta cgcccaggtc tacccggacc tggagctgac cttcgacctc 600

gcgaaggccg acgatttcaa ggcgaagctc ctggatgagc aggccacccc gtccgacaca 660

cagaaggcgc tggtgaacct cctgctctcc agcgatggcg agaaggagat tgtcaagaag 720

aggaagcagg tgctgacaga gttcgcgaag gccatcaccg gcctcaagac aaagttcaac 780

ctcgccctgg gcaccgaggt ggatgaggcc gatgcctcaa attggcagtt ctctatgggc 840

cagctcgacg ataagtggtc caacatcgag acatccatga cagaccaggg caccgagatt 900

ttcgagcaga ttcaggagct gtaccgggcg aggctgctca acggcattgt gccagccggc 960

atgtccctga gccaggccaa ggtcgccgat tacggccagc acaaggagga cctggagctg 1020

ttcaagacat acctcaagaa gctgaatgat catgagctgg cgaagaccat ccggggcctc 1080

tacgacaggt acattaacgg cgacgatgcc aagccgttcc tgcgcgagga cttcgtcaag 1140

gcgctcacca aggaggtgac agcccatcca aacgaggtgt ccgagcagct gctcaacagg 1200

atgggccagg ccaatttcat gctcaagcag aggaccaagg ccaacggcgc aatcccaatt 1260

cagctccagc agcgcgagct ggatcagatc attgcgaacc agagcaagta ctacgactgg 1320

ctcgccgccc caaatccagt ggaggcacac aggtggaaga tgccgtacca gctcgatgag 1380

ctgctcaatt tccatatccc atactacgtc ggccctctga ttacaccgaa gcagcaggcg 1440

gagtcaggcg agaacgtgtt cgcctggatg gtcaggaagg acccgtctgg caacatcacc 1500

ccatacaatt tcgatgagaa ggtggaccgg gaggcgtcag ccaatacatt catccagagg 1560

atgaagacca cagataccta cctgattggc gaggacgtgc tccctaagca gtctctgctc 1620

taccagaagt acgaggtgct caacgagctg aacaatgtcc gcatcaacaa tgagtgcctg 1680

ggcacagatc agaagcagcg gctcatcagg gaggtgttcg agcggcactc atctgtgacc 1740

attaagcagg tcgcggataa tctcgtggca catggcgact tcgcccggag gccggagatc 1800

aggggcctgg cggacgagaa gaggttcctc tccagcctgt ccacatacca ccagctcaag 1860

gagatcctgc atgaggccat tgacgatcca accaagctgc tcgatatcga gaacatcatt 1920

acctggagca cagtgttcga ggaccacacc atcttcgaga caaagctggc cgagattgag 1980

tggctcgacc cgaagaagat caacgagctg tcaggcattc gctaccgggg atggggacag 2040

ttctctcgca agctgctcga cggcctcaag ctgggcaacg gccacacagt cattcaggag 2100

ctgatgctga gcaaccataa tctcatgcag atcctggccg atgagacact caaggagaca 2160

atgacagagc tgaaccagga caagctcaag acagacgata tcgaggatgt gattaacgac 2220

gcgtacacct caccatctaa taagaaggcc ctccggcagg tcctgagggt ggtcgaggat 2280

attaagcatg ccgcgaatgg ccaggaccct tcatggctct tcatcgagac agccgacggc 2340

accggcacag ccggcaagcg cacacagtct cggcagaagc agatccagac cgtgtacgcg 2400

aacgccgcgc aggagctgat tgattccgcc gtgaggggcg agctggagga taagatcgcg 2460

gacaaggcct ctttcacaga ccgcctcgtg ctgtacttca tgcagggcgg cagggatatc 2520

tacacaggcg cccctctgaa tattgaccag ctctcccact acgacatcga tcatattctc 2580

ccgcagtcac tgattaagga cgattctctg gacaacaggg tgctcgtcaa cgcgacaatc 2640

aatcgcgaga agaacaatgt gttcgcctcc acactcttcg cgggcaagat gaaggcaacc 2700

tggcggaagt ggcatgaggc aggcctgatt tcaggacgca agctccggaa tctcatgctg 2760

aggccggatg agatcgacaa gttcgccaag ggattcgtgg caaggcagct ggtggagaca 2820

aggcagatca ttaagctcac agagcagatc gctgccgccc agtaccctaa caccaagatc 2880

attgccgtga aggcgggcct cagccaccag ctgagggagg agctggattt cccgaagaac 2940

agggacgtga atcattacca ccatgcgttc gatgccttcc tcgccgcaag gattggcaca 3000

tacctgctca agcgctaccc gaagctggcg cctttcttca cctacggcga gttcgccaag 3060

gtggacgtga agaagttcag ggagttcaac ttcatcggcg cgctcacaca cgccaagaag 3120

aatatcattg cgaaggatac cggcgagatt gtgtgggata aggagaggga catccgcgag 3180

ctggacagga tctacaattt caagcgcatg ctcattaccc atgaggtcta cttcgagaca 3240

gccgatctct tcaagcagac catctacgcc gcaaaggact ccaaggagag gggcggcagc 3300

aagcagctga ttcctaagaa gcagggctac ccgacacagg tgtacggcgg atacacccag 3360

gagtcaggca gctacaatgc gctcgtgcgc gtggccgagg cagataccac agcctaccag 3420

gtcatcaaga tttccgcgca gaacgcctcc aagatcgcga gcgccaatct caagagcagg 3480

gagaagggca agcagctgct caatgagatt gtggtcaagc agctggcgaa gcgccggaag 3540

aactggaagc cttcagccaa ttctttcaag atcgtgattc cgaggttcgg catgggcaca 3600

ctgttccaga acgccaagta cggcctcttc atggtcaact cagacaccta ctaccgcaat 3660

taccaggagc tgtggctgtc ccgggagaat cagaagctgc tcaagaagct cttcagcatc 3720

aagtacgaga agacacagat gaaccacgat gccctccagg tgtacaaggc catcattgac 3780

caggtcgaga agttcttcaa gctgtacgat atcaatcagt tcagggcgaa gctctccgac 3840

gccattgagc gcttcgagaa gctgccgatc aacaccgatg gcaataagat tggcaagacc 3900

gagacactcc ggcagatcct cattggcctc caggcgaacg gcacaaggtc caacgtgaag 3960

aatctgggca tcaagaccga cctcggcctg ctccaggtcg gcagcggcat caagctcgac 4020

aaggataccc agattgtcta ccagtcaccg tctggcctgt tcaagaggcg catcccactc 4080

gccgacctg 4089

<210> 3

<211> 7511

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgcagtgcag cgtgacccgg tcgtgcccct ctcttgagat aatgagcatt gcatgtctaa 60

gttataaaaa attaccacat attttttttg tcacacttgt ttgaagtgca gtttatctat 120

ctttatacat atatttaaac tttactctac gaataatata atctatagta ctacaataat 180

atcagtgttt tagagaatca tataaatgaa cagttagaca tggtctaaag gacaattgag 240

tattttgaca acaggactct acagttttat ctttttagtg tgcatgtgtt ctcctttttt 300

tttgcaaata gcttcaccta tataatactt catccatttt attagtacat ccatttaggg 360

tttagggtta atggttttta tagactaatt tttttagtac atctatttta ttctatttta 420

gcctctaaat taagaaaact aaaactctat tttagttttt ttatttaata atttagatat 480

aaaatagaat aaaataaagt gactaaaaat taaacaaata ccctttaaga aattaaaaaa 540

actaaggaaa catttttctt gtttcgagta gataatgcca gcctgttaaa cgccgtcgac 600

gagtctaacg gacaccaacc agcgaaccag cagcgtcgcg tcgggccaag cgaagcagac 660

ggcacggcat ctctgtcgct gcctctggac ccctctcgag agttccgctc caccgttgga 720

cttgctccgc tgtcggcatc cagaaattgc gtggcggagc ggcagacgtg agccggcacg 780

gcaggcggcc tcctcctcct ctcacggcac cggcagctac gggggattcc tttcccaccg 840

ctccttcgct ttcccttcct cgcccgccgt aataaataga caccccctcc acaccctctt 900

tccccaacct cgtgttgttc ggagcgcaca cacacacaac cagaactccc ccaaatccac 960

ccgtcggcac ctccgcttca aggtacgccg ctcgtcctcc cccccccccc ctctctacct 1020

tctcaagatc ggcgttccgg tccatggtta gggcccggta gttctacttc tgttcatgtt 1080

tgtgttagat ccgtgtttgt gttagatccg tgctactagc gttcgtacac ggatgcgacc 1140

tgtacgtcag acacgttctg attgctaact tgccagtgtt tctctttggg gaatcctggg 1200

atggctctag ccgttccgca gacgggatcg atttcatgat tttttttgtt tcgttgcata 1260

gggtttggtt tgcccttttc ctttatttca atatatgccg tgcacttgtt tgtcgggtca 1320

tcttttcatg cttttttttg tcttggttgt gatgatgtgg tctggttggg cggtcgttca 1380

agatcggagt agaattaatt ctgtttcaaa ctacctggtg gatttattaa ttttggatct 1440

gtatgtgtgt gccatacata ttcatagtta cgaattgaag atgatggatg gaaatatcga 1500

tctaggatag gtatacatgt tgatgcgggt tttactgatg catatacaga gatgcttttt 1560

gttcgcttgg ttgtgatgat gtggtgtggt tgggcggtcg ttcattcgtt caagatcgga 1620

gtagaatact gtttcaaact acctggtgta tttattaatt ttggaactgt atgtgtgtgt 1680

catacatctt catagttacg agtttaagat ggatggaaat atcgatctag gataggtata 1740

catgttgatg tgggttttac tgatgcatat acatgatggc atatgcagca tctattcata 1800

tgctctaacc ttgagtacct atctattata ataaacaagt atgttttata attattttga 1860

tcttgatata cttggatgat ggcatatgca gcagctatat gtggattttt ttagccctgc 1920

cttcatacgc tatttatttg cttggtactg tttcttttgt cgatgctcac cctgttgttt 1980

ggtgttactt ctgcagcctg caggtagatc gctcgtcgac atgacaaagc tgaaccagcc 2040

atacggcatc ggcctcgata ttggctcaaa ctctatcggc ttcgcggtgg tcgacgccaa 2100

ttcccacctc ctgcgcctga agggcgagac agccatcggc gccaggctct tccgcgaggg 2160

acagtcagcc gcggatcgga ggggctctag gaccacacgg aggcgcctct cccggacaag 2220

gtggcgcctc agcttcctgc gcgacttctt cgcgccacat atcaccaaga ttgaccctga 2280

tttcttcctc cgccagaagt actccgagat cagccctaag gacaaggatc ggttcaagta 2340

cgagaagagg ctgttcaacg accgcaccga tgccgagttc tacgaggatt acccatcaat 2400

gtaccacctg aggctccatc tgatgaccca cacacataag gccgacccga gggagatttt 2460

cctcgccatc caccatattc tcaagtctcg gggccacttc ctcacacctg gagcggccaa 2520

ggatttcaat accgacaagg tggatctgga ggacatcttc ccagccctca cagaggcgta 2580

cgcccaggtc tacccggacc tggagctgac cttcgacctc gcgaaggccg acgatttcaa 2640

ggcgaagctc ctggatgagc aggccacccc gtccgacaca cagaaggcgc tggtgaacct 2700

cctgctctcc agcgatggcg agaaggagat tgtcaagaag aggaagcagg tgctgacaga 2760

gttcgcgaag gccatcaccg gcctcaagac aaagttcaac ctcgccctgg gcaccgaggt 2820

ggatgaggcc gatgcctcaa attggcagtt ctctatgggc cagctcgacg ataagtggtc 2880

caacatcgag acatccatga cagaccaggg caccgagatt ttcgagcaga ttcaggagct 2940

gtaccgggcg aggctgctca acggcattgt gccagccggc atgtccctga gccaggccaa 3000

ggtcgccgat tacggccagc acaaggagga cctggagctg ttcaagacat acctcaagaa 3060

gctgaatgat catgagctgg cgaagaccat ccggggcctc tacgacaggt acattaacgg 3120

cgacgatgcc aagccgttcc tgcgcgagga cttcgtcaag gcgctcacca aggaggtgac 3180

agcccatcca aacgaggtgt ccgagcagct gctcaacagg atgggccagg ccaatttcat 3240

gctcaagcag aggaccaagg ccaacggcgc aatcccaatt cagctccagc agcgcgagct 3300

ggatcagatc attgcgaacc agagcaagta ctacgactgg ctcgccgccc caaatccagt 3360

ggaggcacac aggtggaaga tgccgtacca gctcgatgag ctgctcaatt tccatatccc 3420

atactacgtc ggccctctga ttacaccgaa gcagcaggcg gagtcaggcg agaacgtgtt 3480

cgcctggatg gtcaggaagg acccgtctgg caacatcacc ccatacaatt tcgatgagaa 3540

ggtggaccgg gaggcgtcag ccaatacatt catccagagg atgaagacca cagataccta 3600

cctgattggc gaggacgtgc tccctaagca gtctctgctc taccagaagt acgaggtgct 3660

caacgagctg aacaatgtcc gcatcaacaa tgagtgcctg ggcacagatc agaagcagcg 3720

gctcatcagg gaggtgttcg agcggcactc atctgtgacc attaagcagg tcgcggataa 3780

tctcgtggca catggcgact tcgcccggag gccggagatc aggggcctgg cggacgagaa 3840

gaggttcctc tccagcctgt ccacatacca ccagctcaag gagatcctgc atgaggccat 3900

tgacgatcca accaagctgc tcgatatcga gaacatcatt acctggagca cagtgttcga 3960

ggaccacacc atcttcgaga caaagctggc cgagattgag tggctcgacc cgaagaagat 4020

caacgagctg tcaggcattc gctaccgggg atggggacag ttctctcgca agctgctcga 4080

cggcctcaag ctgggcaacg gccacacagt cattcaggag ctgatgctga gcaaccataa 4140

tctcatgcag atcctggccg atgagacact caaggagaca atgacagagc tgaaccagga 4200

caagctcaag acagacgata tcgaggatgt gattaacgac gcgtacacct caccatctaa 4260

taagaaggcc ctccggcagg tcctgagggt ggtcgaggat attaagcatg ccgcgaatgg 4320

ccaggaccct tcatggctct tcatcgagac agccgacggc accggcacag ccggcaagcg 4380

cacacagtct cggcagaagc agatccagac cgtgtacgcg aacgccgcgc aggagctgat 4440

tgattccgcc gtgaggggcg agctggagga taagatcgcg gacaaggcct ctttcacaga 4500

ccgcctcgtg ctgtacttca tgcagggcgg cagggatatc tacacaggcg cccctctgaa 4560

tattgaccag ctctcccact acgacatcga tcatattctc ccgcagtcac tgattaagga 4620

cgattctctg gacaacaggg tgctcgtcaa cgcgacaatc aatcgcgaga agaacaatgt 4680

gttcgcctcc acactcttcg cgggcaagat gaaggcaacc tggcggaagt ggcatgaggc 4740

aggcctgatt tcaggacgca agctccggaa tctcatgctg aggccggatg agatcgacaa 4800

gttcgccaag ggattcgtgg caaggcagct ggtggagaca aggcagatca ttaagctcac 4860

agagcagatc gctgccgccc agtaccctaa caccaagatc attgccgtga aggcgggcct 4920

cagccaccag ctgagggagg agctggattt cccgaagaac agggacgtga atcattacca 4980

ccatgcgttc gatgccttcc tcgccgcaag gattggcaca tacctgctca agcgctaccc 5040

gaagctggcg cctttcttca cctacggcga gttcgccaag gtggacgtga agaagttcag 5100

ggagttcaac ttcatcggcg cgctcacaca cgccaagaag aatatcattg cgaaggatac 5160

cggcgagatt gtgtgggata aggagaggga catccgcgag ctggacagga tctacaattt 5220

caagcgcatg ctcattaccc atgaggtcta cttcgagaca gccgatctct tcaagcagac 5280

catctacgcc gcaaaggact ccaaggagag gggcggcagc aagcagctga ttcctaagaa 5340

gcagggctac ccgacacagg tgtacggcgg atacacccag gagtcaggca gctacaatgc 5400

gctcgtgcgc gtggccgagg cagataccac agcctaccag gtcatcaaga tttccgcgca 5460

gaacgcctcc aagatcgcga gcgccaatct caagagcagg gagaagggca agcagctgct 5520

caatgagatt gtggtcaagc agctggcgaa gcgccggaag aactggaagc cttcagccaa 5580

ttctttcaag atcgtgattc cgaggttcgg catgggcaca ctgttccaga acgccaagta 5640

cggcctcttc atggtcaact cagacaccta ctaccgcaat taccaggagc tgtggctgtc 5700

ccgggagaat cagaagctgc tcaagaagct cttcagcatc aagtacgaga agacacagat 5760

gaaccacgat gccctccagg tgtacaaggc catcattgac caggtcgaga agttcttcaa 5820

gctgtacgat atcaatcagt tcagggcgaa gctctccgac gccattgagc gcttcgagaa 5880

gctgccgatc aacaccgatg gcaataagat tggcaagacc gagacactcc ggcagatcct 5940

cattggcctc caggcgaacg gcacaaggtc caacgtgaag aatctgggca tcaagaccga 6000

cctcggcctg ctccaggtcg gcagcggcat caagctcgac aaggataccc agattgtcta 6060

ccagtcaccg tctggcctgt tcaagaggcg catcccactc gccgacctgt caagggctga 6120

tcctaagaag aagaggaagg tttctagggc agatccaaag aagaagcgga aggtgtctag 6180

agctgatccg aagaagaagc gcaaggtgtg actcgagata tgaagatgaa gatgaaatat 6240

ttggtgtgtc aaataaaaag cttgtgtgct taagtttgtg tttttttctt ggcttgttgt 6300

gttatgaatt tgtggctttt tctaatatta aatgaatgta agatcacatt ataatgaata 6360

aacaaatgtt tctataatcc attgtgaatg ttttgttgga tctcttctgc agcatataac 6420

tactgtatgt gctatggtat ggactatgga atatgattaa agataagagg atccgcggat 6480

catgaaccaa cggcctggct gtatttggtg gttgtgtagg gagatgggga gaagaaaagc 6540

ccgattctct tcgctgtgat gggctggatg catgcggggg agcgggaggc ccaagtacgt 6600

gcacggtgag cggcccacag ggcgagtgtg agcgcgagag gcgggaggaa cagtttagta 6660

ccacattgcc cagctaactc gaacgcgacc aacttataaa cccgcgcgct gtcgcttgtg 6720

tggagacctt atattcccca gaacatcagg ttaatggcgt ttttgatgtc attttcgcgg 6780

tggctgagat cagccacttc ttccccgata acggaaaccg gcacactggc catatcggtg 6840

gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc acgggagact 6900

ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc gggcgtgtca 6960

ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt ataggtgtaa 7020

accttaaact gcatttcacc agcccctgtt ctcgtcagca aaagagccgt tcatttcaat 7080

aaaccgggcg acctcagcca tcccttcctg attttccgct ttccagcgtt cggcacgcag 7140

acgacgggct tcattctgca tggttgtgct taccagaccg gagatattga catcatatat 7200

gccttgagca actgatagct gtcgctgtca actgtcactg taatacgctg cttcatagca 7260

tacctctttt tgacatactt cgggtataca tatcagtata tattcttata ccgcaaaaat 7320

cagcgcgcaa atacgcatac tgttatctgg cttggtaccg gtctcagtct caggtagatg 7380

tcagatcaat cagaaatgat tgatctgaca tctacgagtt gagatcaaac aaagcttcag 7440

ctgagtttca atttctgagc ccatgttggg ccatacatat gccacccgag tgcaaatcgg 7500

gtggcttttt t 7511

<210> 4

<211> 109

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtctcaggta ggaaactacg agttgagatc aaacaaagct tcagctgagt ttcaatttct 60

gagcccatgt tgggccatac atatgccacc cgagtgcaaa tcgggtggc 109

<210> 5

<211> 153

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gtctcaggta gatgtcagat caatcagttc aaggaaactt gaattgattg atctgacatc 60

tacgagttga gatcaaacaa agcttcagct gagtttcaat ttctgagccc atgttgggcc 120

atacatatgc cacccgagtg caaatcgggt ggc 153

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggcgaacggc acaaggtcca 20

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttctaataaa cgctcttttc tct 23

<210> 8

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggtaccggtc tcagtctcag gtagatgtca gatca 35

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

agtcctggca aacaacctgc agaaa 25

<210> 10

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tggcatttct accttatcga tgaaa 25

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tcccgagcgc ggagtacgta cgaaa 25

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtgtgagtcc tggcaaacaa cctgc 25

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

agacgcaggt tgtttgccag gactc 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gtgtgtggca tttctacctt atcga 25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

agactcgata aggtagaaat gccac 25

<210> 16

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gtgtgtcccg agcgcggagt acgta 25

<210> 17

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agactacgta ctccgcgctc gggac 25

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ttcgcaagta gcagcatcca 20

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

tggtttagtg ggttgttcac tg 22

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

tgatggcata tgcagcagct 20

<210> 21

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

tgtgtgtcgg acggggtggc 20

<210> 22

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Ser Arg Ala Asp Pro Lys Lys Lys Arg Lys Val

1 5 10

Claims (15)

1. A Cas9 protein characterized by:

   (a) the amino acid sequence is shown as SEQ ID No. 1; or:

   (b) the amino acid sequence is the same as SEQ ID NO: 1 has the same or similar biological functions compared with the sequence with one or more amino acid substitutions, deletions or additions.
2. 2. A Cas9 protein according to claim 1, characterized in that: the biological functions are the same or similar, and the biological functions refer to at least one of the following activities:

   an activity of binding to a guide RNA, an activity of binding to a specific site of a target sequence under the guidance of a guide RNA, an endonuclease activity, an activity of binding to a specific site of a target sequence and cleaving a nucleic acid under the guidance of a guide RNA, or a PAM site recognition; further, the PAM site is characterized by any of 5 '-NGAAA-3', N representation A, G, C, T.
3. 3. The Cas9 protein according to claim 1, having an amino acid sequence that is mutated at SEQ ID No.1 by D13A and/or H858A.
4. 4. A gene encoding a Cas9 protein as claimed in any one of claims 1 to 3.
5. 5. The coding gene according to claim 4, characterized in that its nucleotide sequence is represented by Seq ID No. 2.
6. 6. An expression vector comprising the coding gene according to claim 4 or 5.
7. 7. The expression vector of claim 6, which comprises a Cas9 protein expression unit and has the structure of ZmUbi1-LacCas9-NLS-AtHSP, wherein the LacCas9 module is the encoding gene of claim 4 or 5; the ZmUbi1 is a maize Ubi1 promoter, the NLS is a nuclear localization sequence, and the AtHSP is an Arabidopsis HSP terminator.
8. 8. The expression vector of claim 7, wherein the nucleotide sequence of the Cas9 protein expression unit is shown as 1-6467 in Seq ID No. 3.
9. 9. The expression vector of claim 7, further comprising a sgRNA cloning and transcription unit capable of co-expressing the Cas9 protein of any one of claims 1-3 and sgRNAs.
10. 10. The expression vector of claim 7, wherein: the structure of the sgRNA cloning and transcription unit is OsU6-ccdB-sgRNA scaffold-TTTTTT; OsU6 is rice OsU6 promoter; the sgRNA scaffold is an sgRNA framework; the ccdB is an escherichia coli lethal gene; the TTTTTT is a sgRNA transcription termination signal.
11. 11. The expression vector of claim 7, wherein: the nucleotide sequence of the sgRNA scaffold module of the sgRNA cloning and transcription unit is represented by 6477-7512 in Seq ID No.3, Seq ID No.4 or Seq ID No. 5.
12. 12. Use of the expression vector of any one of claims 6 to 11 in construction of a CRISPR-Cas9 gene editing system.
13. A method of CRISPR-Cas9 gene editing characterized in that: the expression vector of any one of 6-11 is used for providing the activity of a Cas9 protein for a CRISPR-Cas9 gene editing system.
14. 14. The method according to claim 13, characterized by the steps of:

    a. constructing a framework vector: constructing the expression vector of any one of claims 6 to 11 as a CRISPR-Cas9 backbone vector;

    b. constructing a directional gene editing vector: designing a gene editing site and sgRNA aiming at a target gene to be edited, synthesizing a primer pair of the sgRNA, annealing the primer pair to form double-stranded DNA with a sticky end, and replacing a ccdB element in a CRISPR-Cas9 skeleton vector with the double-stranded DNA to obtain a directional editing expression vector;

    c. targeted gene editing:

    b, transforming the cell to be edited by using the directional editing expression vector obtained in the step b to obtain a cell or a plant with directional gene editing;

    or;

    b, transforming the protoplast by using the directional editing expression vector obtained in the step b, detecting the gene directional editing condition of the protoplast, and culturing the protoplast successfully subjected to directional editing to obtain a directional gene editing plant;

    or;

    and c, transferring the directional editing expression vector obtained in the step b into Agrobacterium tumefaciens (Agrobacterium tumefaciens), and introducing the vector into a target plant by an Agrobacterium-mediated genetic transformation method to obtain a directional gene editing plant.
15. 15. The method of claim 14, wherein: the step b can be carried out according to the following specific steps:

    a) defining a target DNA region of a plant genome to be edited, analyzing a PAM site characteristic region which can be identified by the Cas9 protein of any one of claims 1-3, and selecting a 20bpDNA sequence adjacent to the 3' end of a PAM structure as a specific modification target sequence; the PAM locus is characterized by 5 '-NGAAA-3', and N represents any one of A, G, C, T;

    b) synthesizing a forward oligonucleotide chain with the characteristics of 5 '-GTGTG-NX-3' and a reverse oligonucleotide chain with the characteristics of 5 '-AGAC-NX-3' according to the selected specific modified target sequence, wherein N represents any one of A, G, C, T, X is an integer, and when the first position of the selected target sequence is G, X is 19; when the first position of the selected target sequence is not G, X ═ 20, where NX in the forward oligonucleotide strand and NX in the reverse oligonucleotide have reverse complementary characteristics; obtaining a complementary oligonucleotide double-stranded fragment by annealing;

    c) and replacing the ccdB element in the CRISPR-Cas9 skeleton vector with the double-stranded DNA to obtain the directional editing expression vector.

Images