CN110747187A

CN110747187A - Cas12a protein for identifying TTTV and TTV double PAM sites, plant genome directed editing vector and method

Info

Publication number: CN110747187A
Application number: CN201911106924.9A
Authority: CN
Inventors: 周建平; 张勇; 郑雪莲; 刘诗诗; 何瑶; 唐旭
Original assignee: University of Electronic Science and Technology of China
Current assignee: University of Electronic Science and Technology of China
Priority date: 2019-11-13
Filing date: 2019-11-13
Publication date: 2020-02-04
Anticipated expiration: 2039-11-13
Also published as: CN110747187B

Abstract

The invention belongs to the field of genetic engineering, and relates to a Cas12a protein for identifying TTTV and TTV double PAM sites, a plant genome directional editing vector and a plant genome editing method. The invention aims to solve the technical problem of establishing a novel CRISPR-Cas12a plant genome directed editing technology capable of efficiently identifying TTTV and TTV double PAM sites. The technical means for solving the technical problems is to provide a novel protein with nuclease Cas12a and a coding gene of the protein. Genome directed editing vectors and editing methods are also provided. The novel Cas12a protein has the ability of recognizing TTTV and TTV double PAM sites. The efficient editing efficiency is achieved at TTTV PAM sites and TTV PAM sites.

Description

Cas12a protein for identifying TTTV and TTV double PAM sites, plant genome directed editing vector and method

Technical Field

The invention belongs to the field of genetic engineering, and relates to a Cas12a protein for identifying TTTV and TTV double PAM sites, a plant genome CRISPR-Cas12a directional editing vector and a plant genome editing method.

Background

The genome directed editing technology is the leading and hot field of biological research, and realizes accurate directed editing of a genome by deleting, replacing, typing in or modifying nucleotides and the like of a specific region of the genome. The genome editing technology mainly comprises the following steps: zinc Finger Nucleases (ZFNs), which achieve gene-targeted manipulation, are widely used. However, the assembly of zinc finger nucleases for ZFNs of specific DNA sequences requires a large amount of work, is very expensive, and has low specificity and efficiency; compared with zinc finger nucleases, transcription activator-like effector nucleases (TALENs) have the advantages of simpler design, higher specificity and more obvious effect. The disadvantages are that the module assembling process is complicated and the cost is high; CRISPR-Cas9(Cluster regulated interspersed short palindromic repeats/CRISPR-associated 9) technology developed in the beginning of 2013 (Sander & Joung). The CRISPR-Cas9 system is widely applied to gene function analysis, new germplasm creation and genetic improvement of animals, plants and microorganisms due to the advantages of simple design and operation, high editing efficiency, wide universality and the like. Although CRISPR-Cas9 is considered to be a revolutionary technology in the field of genetic research, researchers have not abandoned improvements and developments in CRISPR technology. Sequence-specific DNA cleavage mediated by wild-type SpCas9 nuclease requires recognition of the 5 '-NGG-3' PAM site, so that the effective editing interval of SpCas9 nuclease in the target genome is significantly limited. Meanwhile, the SpCas9 genome editing system has potential non-specific cleavage activity, namely 'off-target', and can generate uncertain biological effects on an edited object. To this end, researchers obtained Cas9 nuclease from other different bacteria for gene editing tools, such as SaCas9, StCas9, NmCas9(Cebrian-Serrano & Davies, 2017). Meanwhile, researchers developed Cas9 variants xCas9-NG, SpCas9-NG, VQR-Cas9(NGA PAM), EQR-Cas9(NGAG PAM), VRER-Cas9(NGCG PAM), and SaKKH-Cas9(NNNRRT PAM) that recognize different PAM sequences. Researchers subsequently developed Cas9 variants that could be used in plants.

After the CRISPR-Cas9 system, researchers develop a new type II CRISPR-Cas system-CRISPR-Cas 12a (CRISPR-Cpf1) system, which is different from CRISPR-Cas9 in that the CRISPR-Cas system has the characteristics or advantages that a PAM recognition site of a T/A enrichment region is provided, guide RNA is a single crRNA molecule with a simple structure (can be subjected to self-shearing and processing for maturation), a sticky end is generated, a deleted fragment is relatively large (mostly 6-13bp) and the like, and the CRISPR-Cas system is considered to realize simpler, more accurate and more diversified genome-directed genetic modification. The CRISPR-Cas12a currently used is mainly LbCas12a, AsCas12a and FnCas12 a. Research has shown that: in animal cells, the editing activity of LbCas12a is low at a 4-base PAM recognition site of 'TTTV', the editing activity of AsCas12a and FnCas12a is high at a 4-base PAM site of 'TTTV', but the editing activity of LbCas12a and AsCas12a is inactive at a 3-base PAM site of 'TTV', and the editing activity of FnCas12a is low. LbCas12a, FnCas12a and AsCas12a were reported to have editing activity in plants. LbCas12a has high activity in rice genomes, but LbCas12a nuclease needs to strictly recognize a 4-base PAM site of 'TTTV', so that the effective range of crRNA design in target genomes is limited, and the editing activity in other plants except rice is low or even no activity. FnCas12a has 4-base 'TTTV' as the main PAM recognition site, and has low activity of 3-base 'TTV' and even no editing activity at certain sites. Due to the current lack of diversity of CRISPR-Cas12a sources and the easy generation of off-target effect, the editing efficiency at TTV PAM site is not high. Therefore, the screening and development of a novel efficient CRISPR-Cas12a plant genome directed editing system capable of simultaneously identifying TTTVPAM and TTV PAM sites has important significance.

Disclosure of Invention

The invention aims to solve the technical problem of establishing a novel CRISPR-Cas12a plant genome directed editing technology capable of efficiently identifying TTTV and TTV double PAM sites.

The technical means for solving the technical problems is to provide a novel protein with nuclease Cas12 a. The amino acid sequence of the nuclease Cas12a protein is shown as Seq ID No. 1.

The invention also provides a coding gene of the Cas12a protein.

Further, the nucleotide sequence of the encoding gene of the Cas12a protein is shown as Seq ID No. 2.

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

Wherein, the expression vector contains a Cas12a protein expression unit and has the structure of ZmUbi1-NLS-MorCas12 a-NLS-AtHSP.

Wherein, the ZmUbi1 is a maize Ubi1 promoter. The nucleotide sequence of the promoter of Ubi1 in maize is shown in 1-2003 in SeqID No. 3. The NLS is a nuclear localization sequence. The coding nucleotide sequence of the left NLS is shown in 2004-2054 in Seq ID No.3, and the coding nucleotide sequence of the right NLS is shown in 5808-5858 in Seq ID No. 3. The AtHSP is an Arabidopsis HSP terminator. The nucleotide sequence of the AtHSP coding nucleotide is shown in 5873-6122 in Seq ID No. 3.

Further, the nucleotide sequence of the expression unit of the Cas12a protein in the expression vector is shown as Seq ID No. 3.

Wherein, the expression vector contains a crRNA cloning and transcription unit and can co-express Cas12a protein and crRNA.

Furthermore, the structure of the gRNA cloning and transcription unit is Osubi1-HH Ribozyme-crRNAscope cafmelt-ccdB-HDV Ribozyme-pinII.

Wherein OsUbi1 is rice OsUbi1 promoter, and the nucleotide sequence is shown as 1-1539 in Seq ID No. 4. The HH Ribozyme is an HH Ribozyme, the nucleotide sequence is represented by 1540-1582 in Seq ID No.4, the crRNA scaffold is a crRNA skeleton, and the sequence can be represented by 1583-1603 in Seq ID No.4 or Seq ID No. 5; the ccdB is an Escherichia coli lethal gene, and the coding nucleotide sequence is shown as 1611-2235 in Seq ID No. 4. The AtHSP is an Arabidopsis HSP terminator, and the nucleotide sequence is shown as 2319-2627 in Seq ID No. 4. The HDVRibozyme is an HDV ribozyme, and the nucleotide sequence is shown as 2243-2310 in Seq ID No. 4. The pinII is a pinII gene terminator, and the nucleotide sequence is shown as 2319-2627 in Seq ID No. 4.

Furthermore, the nucleotide sequence of the crRNA cloning and transcription unit in the expression vector is SeqID No.4 or Seq ID No. 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-Cas12a gene editing system.

The invention also provides a CRISPR-Cas12a gene editing method. The method uses the expression vector to provide the activity of the Cas12a protein for the CRISPR-Cas12a gene editing system.

Further, the method comprises the following steps:

a. constructing a framework vector: the expression vector is used as a CRISPR-Cas12a skeleton vector;

b. constructing a directional gene editing vector: designing a gene editing site and crRNA aiming at a target gene to be edited, artificially synthesizing a primer pair of the crRNA, annealing the primer pair to form double-stranded DNA with a sticky end, and replacing a ccdB element in a CRISPR-Cas12a skeleton vector with the double-stranded DNA to obtain a directional editing expression vector;

c. editing and detecting directional genes: 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, 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) defining a target DNA region of a plant genome to be edited, analyzing a PAM site characteristic region which can be identified by the Cas12a nuclease protein, and selecting a 23bpDNA sequence adjacent to the 3' end of the PAM structure as a specific modification target sequence; any one of the PAM site features 5 '-TTV-3' and/or 5 '-TTTV-3', V represents A, G, C;

b) synthesizing a forward oligonucleotide chain with the characteristics of 5 '-AGAT-NX-3' and a reverse oligonucleotide chain with the characteristics of 5 '-GGCC-NX-3' according to the selected specific modified target sequences, wherein N represents any one of A, G, C, T, X is an integer, and 18 ≦ X ≦ 25, preferably X ≦ 23, wherein NX in the forward oligonucleotide chain 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-Cas12a 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 Cas12a protein, and the Cas12a protein has the capacity of recognizing TTTV and TTV double PAM sites. Through the codon optimization of the Cas12a protein gene, the protein coding gene suitable for plant CRISPR-Cas12a gene editing is obtained. On the basis, a novel efficient CRISPR-Cas12a plant genome directed modification skeleton vector is also provided, and the plant genome directed modification can be simply, quickly and efficiently carried out. Experiments show that the CRISPR-Cas12a skeleton vector has high-efficiency editing efficiency on all detected TTTV PAM sites and high-efficiency editing activity on all detected TTV PAM sites, is a novel system with double PAM site high-efficiency editing capacity, and has good application prospects.

Drawings

Fig. 1 electropherogram of homologous gene clone of MorCas12 a. M represents DNA molecule marker, lane 1 represents sample PCR product electrophoresis, and arrow represents PCR amplification product

FIG. 2 is a schematic structural diagram of a MorCas12a plant genome directed modification backbone vector used in an embodiment of the present invention.

a. MorCas12 a-crRNA-scaffold 1, MorCas12 a-crRNA-scaffold 2, MorCas12 a-crRNA-scaffold 3, and MorCas12 a-crRNA-scaffold 4;

b. MorCas12 a-crRNA-scaffold 5, MorCas12 a-crRNA-scaffold 6, MorCas12 a-crRNA-scaffold 7, and MorCas12 a-crRNA-scaffold 8.

Figure 3 shows in protoplasts the results of the experiments on the editing activity of MorCas12 on three TTTV PAM sites.

Figure 4 shows in protoplasts the results of the experiments on the editing activity of MorCas12 on three TTV PAM sites.

Detailed Description

In order to obtain a CRISPR-Cas12a system with better performance that can meet the various needs in the art, the inventors performed a great deal of search and validation work on a new Cas12a prior to the present application. In a microorganism sample containing human body source, some possible Cas12 encoding genes are obtained in a homologous gene cloning mode, and preliminary functional verification is carried out. Among them, a novel gene was found which encodes a protein having the activity of a typical CRISPR-Cas12a protein. More unexpectedly, the protein encoded by the gene not only has high-efficiency editing activity on TTTV PAM sites, but also has high-efficiency editing activity on all TTV PAM sites. Through further studies, it was confirmed that the similarity was 99.84% to the Moraxella bacteria Moraxella bovoculi. CRISP Cas12a protein sequence (numbered as WP _046697655.1 in NCBI), 95.57% to the Moraxella caprae CRISP Cas12a protein sequence (numbered as WP _036388671.1 in NCBI) and 95.18% to the Moraxella bovis CRISP Cas12a protein sequence (numbered as WP _115369192.1 in NCBI). Therefore, the CRISPR-Cas12a gene is named as MoraxellaspCRISPR-Cas12a (MorCRISPR-Cas 12a for short).

Furthermore, according to the genome structure and expression characteristics of plants, the plant genome directional modification skeleton vector based on the MorCRISPR-Cas12a gene is constructed, so that the effective application of the gene editing system based on the MorCRISPR-Cas12a in the plant genome directional modification is realized.

The CRISPR-Cas12a provided by the invention is mainly composed of two parts as a skeleton vector of a gene editing tool, wherein one part is used for expressing Cas12a protein which plays a role in cutting DNA, and the other part is used for expressing crRNA which plays a role in guiding, namely directing. There are two major factors that determine editing power: the first is the cleavage capacity of Cas12a protein and the expression quantity thereof in cells; second is crRNA scaffold and its expression level.

First, to ensure the expression level of the MorCas12a protein. The maize Ubi1 promoter disclosed in the reference document (Tang X et al. A CRISPR-Cpf1system for effective gene expression and translational expression. nat Plants, 2017,3:17103.) can efficiently promote the expression of CRISPR-Cpf 1and record gene editing. Constructing a ZmUbi1 promoter-initiated MorCas12 nuclease protein expression unit

(ZmUbi1-NLS-MorCas12 a-NLS-AtHSP). Firstly, the MorCRISPR-Cas12 gene is subjected to codon optimization according to the codon characteristics of the plant gene, and then the MorCas12a nuclease protein (namely NLS-MorCas12a-NLS) containing NLS (nuclear localization sequence) coding sequences at the 5 'end and the 3' end is synthesized, wherein the coding nucleotide sequence is shown in 2004-3858 in Seq ID No. 3. Then the NLS-MorCas12a-NLS unit, the constitutive promoter ZmUbi1 element from maize (DNA sequence shown as 1-2003 in Seq ID No. 3) and the Arabidopsis AtHSP terminator element (DNA sequence shown as 5873-6122 in Seq ID No. 3) were assembled into a MorCas12 nuclease protein expression unit by the Gibson Assembly technology.

Secondly, the transcription of crRNA into the expression of crRNA scaffold in the cloned unit may also play an important role in the editing effect. The present reference (Tang X et al. A CRISPR-Cpf1system for effective genetic engineering and translational expression in plantations. nat plantations, 2017,3:17103.) describes that the maize Ubi1 promoter is capable of promoting the expression of crRNA. The rice Ubi1 promoter was used to initiate crRNA transcription. Because the crRNA scaffold structure has important influence on the editing activity, the invention selects four different crRNAs cafpold: scaffold1 derived MorCas12a nuclease; the expression clone unit of crRNA transcription is obtained by respectively assembling a scaffold2 which is a document from 4N96 (Teng Fet. al. enhanced mammalian Genome edition by New Cas12a orthogonal with optimized crRNA scaffolds, Genome biol.2019Feb 5; 20(1) 15; scaffold3 derived from LbCas12a nuclease and scaffold4 derived from FnCas12a nuclease), and has the structure of Osubi1-HH Ribozyme-crRNA scaffold-ccdB-HDV Ribozyme-pinII).

The following specific configurations are listed in the examples section of the present invention: a rice OsUbi1 promoter (nucleotide sequence is shown as 1-1539 in Seq ID No. 4), a synthetic sequence HHRibozyme-crRNA scaffold-ccdB-HDV Ribozyme unit (nucleotide sequence is shown as 1540-2318 in Seq ID No.4 or Seq ID No. 5) and a pinII terminator (nucleotide sequence is shown as 2319-2627 in Seq ID No. 4) are assembled into a crRNA transcription expression cloning unit OsUbi1-HH Ribozyme-crRNA scaffold-ccdB-HDV Ribozyme-pinII by a Gibson Assembly technology.

Wherein the HH Ribozyme-crRNA scaffold-ccdB-HDV Ribozyme unit is also designed with a BsaI-ccdB-BsaI sequence having a nucleotide sequence as shown in 1611-2235 in Seq ID No. 4. The function of the recombinant plasmid is to be used as a multiple cloning site to perform enzyme digestion on a MorCRISPR-Cas12a oriented modification framework vector so as to clone a target guide crRNA specific target sequence (protospacer) onto the framework vector.

Meanwhile, the examples of the present invention also report that OsU6 promoter can effectively start crRNA, redesign and construct directional editing backbone vector of MorCRISPR-Cas12a containing MorCas12a nuclease protein expression unit ZmUbi1-NLS-MorCas12a-NLS-AtHSP unit and OsU6 promoter start crRNA transcription expression unit OsU6-crRNA scaffold-ccdB-ploy (T) according to the literature (Endo A et al, effective targeted mutagenesis of rice and tobaco genetics using Cpf1from Franciella novicida. Sci Rep,2016,6: 38169).

The specific construction process is as follows: first, the MorCas12a nuclease protein expression unit ZmUbi1-NLS-MorCas12a-NLS-AtHSP as described above was prepared.

Secondly, OsU6 promoter was constructed to promote the transcription of crRNA expression unit OsU6-crRNA scaffold-ccdB-ploy (T): the rice OsU6 promoter (see published backbone vector pZHY988 for sequence) was assembled into crRNA transcriptional expression cloning units by Gibson Assembly technology, synthesizing four different crRNA scaffold units (described supra). .

The nucleotide sequence of BsaI-ccdB-BsaI in each of the above-mentioned backbone vectors is shown by 1611-2235 in Seq ID No. 4. ploy (T) is 10 Ts.

Finally, the new MorCRISPR-Cas12a plant genome directed functional units, namely a MorCas12 nuclease protein expression unit and a crRNA transcription expression cloning unit are cloned into a framework vector pTrans _210d (sequences such as sequence shown in the specification) by a Golden Gate technologyA Multipurpose Toolkit to Enable Advanced genome engineering in plants plant Cell,2017,29(6): 1196-.

By using the Cas12a protein and CRISPR-Cas12a plant genome directed editing framework vector, and combining with a specific genome editing method reported in the field, the directed genome editing aiming at TTTV PAM sites and/or TTVPAM sites can be conveniently carried out in plants. The present invention is further illustrated by the following detailed description of examples.

Example 1 homologous cloning of MorCRISPR-Cas12a

Using a sample containing a human-derived microorganism as a template, carrying out PCR amplification by using degenerate primers, and displaying the result by agarose electrophoresis (FIG. 1): nonspecific bands of approximately 4000bp (target band), 800bp and 300bp were obtained. And (3) cutting and recovering a target band, cloning TA, converting competent cells, selecting a single colony, carrying out PCR identification and sequencing, and finally screening to obtain a novel CRISPR-Cas12 gene with the activity of a typical CRISPR-Cas12a protein. Sequence alignment in NCBI showed that it was 99.84% similar to Moraxella bacteria Moraxella bomboculi. CRISP Cas12a protein sequence (numbering: WP _046697655.1 in NCBI), 95.57% similar to Moraxella caprae CRISP Cas12a protein sequence (numbering: WP _036388671.1 in NCBI) and 95.18% similar to Moraxella bombvis CRISP Cas12a protein sequence (numbering: WP _115369192.1 in NCBI). Therefore, the CRISPR-Cas12a gene is named as MoraxellaspCRISPR-Cas12a (namely, MorCRISPR-Cas12 a).

Example 2 construction of MorCRISPR-Cas12a plant genome directed modification backbone vector

1. A new MorCRISPR-Cas12a gene editing skeleton vector is constructed by adopting different module assembly modes.

Firstly, module 1(MOD _ A) was designed as a MorCas12 nuclease protein expression unit (ZmUbi1-NLS-MorCas12a-NLS-AtHSP) containing NLS coding sequences at the 5 'end and the 3' end, wherein the MorCas12a was synthesized by a bio-company after codon optimization and submitted to the company NLS-MorCas12a-NLS sequence as shown in Seq ID No.3, 2004-6158, and then the NLS-MorCas12a-NLS unit, a constitutive promoter ZmUbi1 element from maize (nucleotide sequence as shown in 1-2003 in Seq ID No. 3) and an Arabidopsis thaliana AtHSP terminator element (nucleotide sequence as shown in 5873-6122 in Seq ID No. 3) were assembled into module 1 by Gibsonassably technology. Next, module 2(MOD _ B) was designed as the crRNA transcriptional expression cloning unit OsUbi1-HH Ribozyme-crRNA scaffold-ccdB-HDV Ribozyme-pinII. The rice OsUbi1 promoter (nucleotide sequence shown as 1-1539 in Seq ID No. 4), synthetic sequences such as the nucleotide sequence shown as 1540-2318 in Seq ID No.4 or Seq ID No.5, and the pinII terminator (nucleotide sequence shown as 2319-2627 in Seq ID No. 4) were assembled into Module 2 by the Gibson Assembly technique. Finally, Module 1, Module 2 was assembled into pTrans _210d by Golden Gate method (sequences such as

Etc., A Multipurposide Toolkit to Enable Advanced genoengineering in plants Cell,2017,29(6): 1196-. Similarly, scaffold3 (with sequence as shown in Seq ID No. 6) and scaffold4 (with sequence as shown in Seq ID No.7) were assembled into expression units, respectively. New MorCRISPR-Cas12a gene editing framework vectors, such as MorCas12a-scaffold1, MorCas12a-scaffold2, MorCas12a-scaffold3 and MorCas12a-scaffold4, are obtained finally through transforming bacterial competence, monoclonal PCR verification, recombinant plasmid extraction, Sanger sequencing verification (FIG. 2 a).

2. Meanwhile, according to the literature (Endo A, Masafumi M, Kaya H, Toki S. efficient targeted transcription of the rice and tobacaco genes using Cpf1from Franciselanovicid. Sci Rep.2016,6:38169), a gene construct comprising a MorCas12a nuclease protein expression unit (ZmUbi1-NLS-MorCas12a-NLS-AtHSP unit (same sequence as module 1) and a module 3(MOD _ C) OsU6 promoter initiates crRNA transcription of the expression unit OsU6-DR-crRNA scaffold-ccdB-DR-ploy (T). Rice OsU6 promoter (see htnshink/www.addgene.org/search/advanced transcription/. pZq 8) was assembled by Gibsonassembe technology into a nucleotide sequence of the gene clone 210, and finally the gene construct a gene construct by the gene construct into a gene construct such a gene construct as a gene construct module by transforming the gene into a gene sequence of the gene construct cDNA clone No.5, monoclonal PCR verification, recombinant plasmid extraction, Sanger sequencing verification and finally obtaining new MorCRISPR-Cas12a gene editing skeleton vectors of MorCas12a-scaffold5, MorCas12a-scaffold6, MorCas12a-scaffold7 and MorCas12a-scaffold8 (figure 2 b).

Example 3 construction of Oryza sativa endogenous Gene Targeted modification based on MorCRISPR-Cas12a System

1. Design of rice endogenous gene guided crRNA

To test the editing activity of the constructed CRISPR-Cas12a system, the rice genome was scanned to obtain the same crRNA sites of PAM site, TTV and TTTV respectively (see table 1) to design crRNA. The corresponding forward and reverse oligonucleotide chains were synthesized artificially based on the designed nucleic acid sequence of crRNA site (the upper case base sequence represents the designed site-specific guide crRNA site; the lower case base sequence represents the cohesive end complementary to the backbone vector) (see Table 1)

TABLE 1 CrRNA design sites, sequences

Cas12a + crRNA recombinant expression vector construction

Respectively mixing the crRNA01-F/R, the crRNA02-F/R and the crRNA03-F/R in equal proportion, boiling in a water bath for 10min, and then naturally cooling and annealing to form double-stranded DNA with a sticky end as an insert for constructing a recombinant vector. Adding different CRISPR-Cas12a plant genome directional modification skeleton vectors, sticky end insertion fragments, BsaI endonuclease and T4DNA ligase into a 200uL PCR tube, carrying out enzyme digestion and connection at 37 ℃ for 5min → 16 ℃ for 10min (15 cycles) → 37 ℃ for 10min → 80 ℃ for 10min → 4 ℃ for 10min, and taking reaction products for carrying out escherichia coli transformation. Positive transformants were identified by kanamycin resistance screening, colony PCR and enzyme digestion, and finally a new recombinant expression vector was obtained by sequencing validation:

MorCas12a-scaffold1-crRNA01、MorCas12a-scaffold1-crRNA02、

MorCas12a-scaffold1-crRNA03、MorCas12a-scaffold2-crRNA01、

MorCas12a-scaffold2-crRNA02、MorCas12a-scaffold2-crRNA03、

MorCas12a-scaffold3-crRNA01、MorCas12a-scaffold3-crRNA02、

MorCas12a-scaffold3-crRNA03、MorCas12a-scaffold4-crRNA01、

MorCas12a-scaffold4-crRNA02、MorCas12a-scaffold4-crRNA03、

MorCas12a-scaffold5-crRNA01、MorCas12a-scaffold5-crRNA02、

MorCas12a-scaffold5-crRNA03、MorCas12a-scaffold6-crRNA01、

MorCas12a-scaffold6-crRNA02、MorCas12a-scaffold6-crRNA03、

MorCas12a-scaffold7-crRNA01、MorCas12a-scaffold7-crRNA02、

MorCas12a-scaffold7-crRNA03、MorCas12a-scaffold8-crRNA01、

MorCas12a-scaffold8-crRNA02、MorCas12a-scaffold8-crRNA03。

rice protoplast transformation of CRISPR-Cas12a recombinant expression vector

The specific procedures for isolating and transforming Nipponbare protoplasts of rice using the above CRISPR-Cas12a recombinant expression vector were carried out by the method disclosed in the reference literature (Tang X, Zheng X, Qi Y, Zhang D, Cheng Y, Tang A, Voytas DF, Zhang Y.2006.A Single Transcript CRISPR-Cas9 System for Efficient Genome expression in plants. mol Plant,9(7): 1088-.

4. Detection of directional modification results

After transformation of rice protoplast, dark culture is carried out for 48 hours at 25 ℃, transformed cells are collected, rice protoplast genome DNA is extracted by a CTAB method, PCR amplification and restriction endonuclease verification analysis are carried out by taking the DNA as a template, and used PCR primers and restriction endonuclease are shown in Table 2. Specific Experimental procedures disclosed in the literature references (Zhong Z, Zhang Y, You Q, Tang X, RenQ, LiuS, Yang L, Wang Y, Liu X, Liu B, Zhang T, Zheng X, Le Y, Zhang Y, Qi Y.2018.molecular Plant. Plant genome editing using FnCpf1and Lbppf 1 nucleic acids at refined and modified PAM sites. mol Plant,11: 999-. After PCR digestion electrophoresis, the value of each band was calculated by software (Image Lab 3.0, Bio-RAD), and the mutation efficiency was the percentage of the sum of the digestion band value and the resistance band value occupied by the resistance band value.

TABLE 2PCR-RZ primer sequences, products and endonuclease information

As can be seen from fig. 3: among the selected TTTV PAM sites, MorCas12a-scaffold1, MorCas12a-scaffold2, MorCas12a-scaffold3, and MorCas12a-scaffold4 all have higher editing activity at all three sites of crRNA01, crRNA02, and crRNA03, and the activities of MorCas12a-scaffold 1and MorCas12a-scaffold2 are preferably close to 60% at the highest and more than 30% at the lowest, which is equivalent to the activity of control LbCas12a (used as a CRISPR-Cpf1system for facilitating gene editing and describing textual expression in Plants. Nat Plants, 2017,3: 17103). Whereas the editing activity of MorCas12a-scaffold5, MorCas12a-scaffold6, MorCas12a-scaffold7 and MorCas12a-scaffold8 at three sites is less than 15%, the editing activity of FnCas12a (the used skeleton vector is shown in Zhong Z, Zhang Y, You Q, Tang X, Ren Q, Liu S, Yang L, Wang Y, Liu X, Liu B, Zhang T, ZHEN X, LeY, Zhang Y, Qi Y.2018.molecular Plant.

As can be seen from fig. 4: among the selected TTV PAM sites, MorCas12a-scaffold 1and MorCas12a-scaffold2 had very high editing activity at all three sites of crRNA01, crRNA02 and crRNA03, with an average activity of over 40%, while MorCas12a-scaffold3, MorCas12a-scaffold4, MorCas12a-scaffold5, MorCas12a-scaffold6, MorCas12a-scaffold7, MorCas 6312 12a-scaffold8 had editing activity at all three sites of less than 25%, and LbCas12a had no editing activity at all sites, FnCas12a had high editing activity at the sites of crRNA01 and crRNA02, but had very low editing activity at the site of crRNA 03. In conclusion, the MorCas12a gene editing system of the present invention has the best editing activity at the TTV PAM recognition site.

In conclusion, the CRISPR-Cas12a gene editing system (especially the MorCas12a-scaffold 1and the MorCas12a-scaffold 2) can not only maintain high editing activity at a TTTV PAM recognition site, but also have high editing activity at a TTVPAM recognition site. Widens the selection of plant genome editing sites and provides a high-efficiency plant genome editing tool.

Claims

Cas12a protein, characterized in that its amino acid sequence is shown as Seq ID No. 1.
2. A gene encoding Cas12a protein as set forth in claim 1.
3. The gene encoding Cas12a protein according to claim 2, characterized in that the nucleotide sequence is shown as Seq id No. 2.
4. An expression vector comprising the coding gene according to any one of claims 2 or 3.
5. The expression vector of claim 4, wherein: the expression vector contains a Cas12a protein expression unit and has the structure of ZmUbi1-NLS-MorCas12 a-NLS-AtHSP.
6. The expression vector of claim 5, wherein: the nucleotide sequence of the Cas12a protein expression unit is shown as Seq ID No. 3.
7. The expression vector of claim 5, wherein: also contains a crRNA clone and a transcription unit, and can co-express Cas12a protein and crRNA.
8. The expression vector of claim 5, wherein: the structure of the crRNA cloning and transcription unit is Osubi1-HH Ribozyme-crRNA scaffold-ccdB-HDV Ribozyme-pinII.
9. The expression vector of claim 5, wherein: the nucleotide sequence of the crRNA cloning and transcription unit is described as Seq ID No.4 or Seq ID No. 5.
10. The expression vector of claim 5, wherein: also contains a hygromycin resistance selection gene.
11. Use of the expression vector of any one of claims 4 to 10 in construction of a CRISPR-Cas12a gene editing system.
A method of CRISPR-Cas12a gene editing characterized in that: the expression vector of any one of claims 4 to 12 is used for providing the activity of a Cas12a protein for a CRISPR-Cas12a gene editing system.
13. The method according to claim 12, characterized by the steps of:

a. constructing a framework vector: constructing the expression vector of any one of claims 4 to 12 as a CRISPR-Cas12a backbone vector;

b. constructing a directional gene editing vector: designing a gene editing site and crRNA aiming at a target gene to be edited, artificially synthesizing a primer pair of the crRNA, annealing the primer pair to form double-stranded DNA with a sticky end, and replacing a ccdB element in a CRISPR-Cas12 skeleton vector with the double-stranded DNA to obtain a directional editing expression vector;

c. editing and detecting directional genes: 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, and introducing the vector into a target plant by an agrobacterium-mediated genetic transformation method to obtain a directional gene editing plant.