CN105543196A - Plant Cas9 variant protein VRER as well as encoding gene and application thereof - Google Patents

Abstract

The invention discloses plant Cas9 variant protein VRER. An amino acid sequence of the protein is shown as SEQ ID No: 2. The invention also discloses a plant Cas9 variant gene VRER. The gene encodes the plant Cas9 variant protein VRER, and an amino acid sequence of the gene is shown as SEQ ID No: 1. The invention further discloses a recombinant expression vector containing the gene. The plant Cas9 variant protein VRER has the application that a genome editing function is realized under the condition that the PAM (protospacer adjacent motif) region is 5'-NGCG-3'.

Description

Plant Cas9 variant protein VRER and its coding gene and application

technical field

The invention relates to the field of biotechnology, in particular to the application of the Cas9 protein variant VRER and its coding gene for editing plant genes.

Background technique

In recent years, CRISPR-Cas9 has rapidly become the most important genome editing method in plants due to its high-efficiency and simple technical characteristics. CRISPR-Cas was originally an immune system widely distributed in prokaryotes. CRISPRs (Clustered regularly interspaced short palindromic repeats) are regularly clustered interspaced short palindromic repeats. Cas protein is a nuclease guided by RNA.

The currently used CRISPR-Cas9 system is a genome editing technology developed based on type II CRISPR-Cas. The Cas protein involved in this technology is only Cas9, which is guided by the guide RNA composed of tracrRNA and crRNA (later optimized as sgRNA). 3-8bp double-strand cut and cause double-strand breaks. When a double-strand break occurs, the cell will initiate two sets of repair mechanisms, namely non-homologous end-joining (Non-homologous ending-joining, NHEJ) and homologous recombination (Homologous recombination, HR). Non-homologous end joining usually results in single or multiple base deletions and insertions at double-strand breaks, and individuals repaired in this way often produce site-directed mutations in a gene. Homologous recombination uses homologous sequences as templates for high-fidelity repair.

In the CRISPR/Cas9 system, the protospace radjacent motif (PAM) sequence determines the distribution of target sequences throughout the genome. PAM is a sequence located near the target DNA and has the function of recognizing the target DNA. Only when the PAM sequence is correctly combined can the two endonuclease active regions of Cas9 be activated. The trinucleotide region PAM (5'-NGG-3') at the end of the target sequence is the recognition site of Cas9, which is the key to realize the cutting function. However, this does not fully meet people's needs for the scope of plant gene editing. Since the selection of target sites of the CRISPR-Cas9 system is limited by the PAM sequence NGG, it is very important to discover and engineer Cas9 variants that can recognize new PAMs in plants.

Contents of the invention

The technical problem to be solved by the present invention is to provide a novel plant Cas9 variant protein VRER and its encoding gene and application.

In order to solve the above technical problems, the present invention provides a plant Cas9 variant protein VRER, the amino acid sequence of which is shown in SEQ ID No: 2.

As an improvement of the plant Cas9 variant protein VRER of the present invention: the protein also includes derivatives generated by adding, substituting, inserting and deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO:2.

The present invention also provides a plant Cas9 variant gene VRER, which encodes a plant Cas9 variant protein VRER, and the nucleotide sequence of the gene is shown in SEQ ID NO:1.

As an improvement of the plant Cas9 variant gene VRER of the present invention: the gene also includes mutants produced by adding, substituting, inserting and deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 1, etc. genes and derivatives.

The present invention also provides a recombinant expression vector comprising the above-mentioned gene.

The present invention also provides the use of the above-mentioned plant Cas9 variant protein VRER: it can realize genome editing function under the condition that the PAM region is 5'-NGCG-3'. N stands for any nucleotide of A, T, C, G.

The scheme of the present invention is specifically as follows:

The protein provided by the present invention is as follows (a) or (b):

(a) a protein consisting of the amino acid sequence shown in Sequence 2 in the Sequence Listing;

(b) A protein derived from Sequence 2 by substituting and/or deleting and/or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2 in the Sequence Listing and having the same activity and function.

In the above protein, the substitution and/or deletion and/or addition of one or several amino acid residues refers to the substitution and/or deletion and/or addition of no more than ten amino acid residues.

Genes encoding the above proteins are also within the protection scope of the present invention.

Above-mentioned gene is the DNA molecule described in any one of following (1)-(3):

1) DNA sequence of sequence 1 in the sequence listing;

2) A polynucleotide encoding the protein sequence of Sequence 2 in the Sequence Listing;

3) A DNA sequence that has more than 90% homology with the DNA sequence defined in Sequence 1 in the sequence listing and encodes the same functional protein;

Recombinant vectors containing any of the above genes also belong to the protection scope of the present invention, such as recombinant expression vectors.

An existing plant expression vector can be used to construct a recombinant expression vector containing the gene.

The plant expression vectors include binary Agrobacterium vectors (such as pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300, etc.) and vectors that can be used for plant microprojectile bombardment.

The application of the above-mentioned proteins, above-mentioned genes or above-mentioned recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria in editing plant genomes is also within the protection scope of the present invention.

In the present invention, a gene encoding a novel Cas9 variant protein VRER is produced by mutating the gene encoding Cas9, and the recognized PAM is NGCG. In the experiment of using NGCG as the PAM sequence to screen the target site and edit the GL1-1 gene in rice, the mutant plants with GL1-1 function loss were successfully obtained through transgenic means, indicating that the protein can recognize 5'-NGCG as the PAM sequence and Make edits to the target site. The invention has broad application prospects in expanding the scope of plant gene editing.

The application of the present invention is to perform gene editing on a specific target site of plant genome, that is, the PAM region is the target site of 5'-NGCG-3'. As a member of the gene editing technology CRISPR-Cas9 system, compared with the limitation that the traditional Cas9 protein can only edit the target site of the 5'-NGG-3' PAM region, it expands the editable range of the system in the plant genome. scope.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 is a schematic diagram of some elements and mutation sites of the recombinant vector.

Figure 2 shows the results of GL1-1 target site enzyme digestion detection; M is marker; 1, 5, 6, 7, 11, 12, 15, 16, 17 are knockout heterozygous plants; 14 is knockout homozygous plants; The last two lanes are enzyme-digested and non-enzyme-digested wild-type respectively; the rest of the lanes are knockout-negative plants, and the enzyme-digested results are no different from the enzyme-digested wild-type.

Figure 3 shows the sequencing results of the GL1-1 target site; the underlined wavy line indicates the target site; the single underline indicates the PAM sequence; the frame indicates the enzyme recognition site; deletions are indicated by short bars, and insertions are indicated by Indicated by lowercase letters.

Figure 4 shows the phenotypes of wild-type Zhonghua 11 and mutant GL1-1.

Figure 5 shows the sequencing results of the LG1 target site; the underlined wavy line indicates the target site; the single underline indicates the PAM sequence; the box indicates the enzyme recognition site; deletions are indicated by short bars, and insertions are indicated by lowercase letters express.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the acquisition of Cas9 variant VRER

Mutations were introduced at specific sites in the Cas9-encoding gene by overlap-extension PCR. The complete Cas9 needs to be divided into 4 segments with the 1135th amino acid, 1218th amino acid and 1336th amino acid as the boundary, and then its coding sequence is amplified by PCR. The primers used are shown in Table 1; KODFX DNA polymerase (purchased from Toyobo Biotechnology Co., Ltd.) PCR amplified the target band, and the reaction conditions were as follows:

Remarks: pC1300-Cas9 can come from application number 201510485573.2.

Reaction program: 94°C, denaturation for 2 minutes; then denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 68°C (1000bp/min), 35 cycles of amplification; finally extension at 68°C for 5 minutes.

Sections 1, 2, 2, 3 and 3, 4 contain a 20bp overlapping region; the first section and the fourth section contain regions homologous to the upper and lower reaches of the vector, respectively. At the beginning of primer design, specific mutations were included in the overlapping portion of the two fragments. The overlapping portion of the reverse primer of Fragment 1 and the forward primer of Fragment 2 contains the codon for amino acid "V", replacing the original amino acid "D" at position 1135. The overlapping portion of the reverse primer of Fragment 2 and the forward primer of Fragment 3 contains the codon for amino acid "R", replacing the original amino acid "G" at position 1218. The overlapping portion of the reverse primer of Fragment 3 and the forward primer of Fragment 4 contains codons for amino acids "Q" and "R", replacing the original amino acid "R" at position 1335 and amino acid "T" at position 1337, respectively. The forward primer of Fragment 1 and the reverse primer of Fragment 3 respectively contain sequences homologous to the upstream and downstream of the vector. Subsequently, through multi-fragment recombination technology, several amplified fragments were overlapped and spliced into a complete Cas9 variant VRER coding sequence. The multi-fragment recombination kit was purchased from Nanjing Novizan Biotechnology Co., Ltd. The vector pC1300-Cas9 was linearized by double enzyme digestion with AatII and SpeI (purchased from NEB Company). The double enzyme digestion reaction system is as follows:

Digestion at 37°C for 3 hours, followed by purification with the Biomed Gel Extraction Kit (Biomed, DR0103) according to the product instructions; the fragment pC1300-Cas9/AatII+SpeI was obtained.

The recombination reaction system is as follows:

Placed at 37°C for 30 minutes. After the reaction was completed, the reaction tube was immediately cooled in an ice-water bath for 5 min. The product was transformed into Escherichia coli competent cell DH5α to obtain the recombinant plasmid pC1300-VRER, and the recombinant plasmid was sequenced to confirm that the mutation was introduced correctly. That is, the recombinant plasmid pC1300-VRER contains the gene shown in SEQ ID NO:1.

instruction manual:

SEQ ID NO: 1 is the VRER DNA sequence; the underline is the nuclear localization signal region (NLS); the uppercase is the stop codon; the rest is the VRER protein coding sequence, and the transformation site counting starts from the VRER protein coding sequence.

SEQ ID NO: 2 is the corresponding VRER protein sequence; the underline is the nuclear localization signal region (NLS); the rest is the VRER protein sequence, and the transformation site counting starts from the VRER protein coding sequence.

Amplification primers of each fragment in table 1

fragment forward primer reverse primer Fragment 1 cgcctctcggattacgacgtcgatca caccggtgtgagggaactagtttga Fragment 2 gttggtgacacgaaacctccatactt tggagctcgcgtgcggaagccaacat Fragment 3 cttccgcacgcgagctccagaagggt ttcgtagagcggtactccttccgatcaatcgt Fragment 4 atcggaaggagtaccgctctacgaaggaggtg caccggtgtgagggaactagtttga

Example 2, Design of GL1-1 Gene sgRNA and Construction of Recombinant Plasmid

The GL1-1 gene sequence "5'-ACGTTGCAGTGGC CCATGG CGCG" was selected as the target sequence, "CGCG" was the PAM sequence, and the underline was the NcoI digestion recognition site. Synthesize positive and negative two oligonucleotide chains, as shown in Table 2. Mix 20 μL of 100 μM g++ and g-- primers (i.e., oligonucleotide primers (5’-3’) in Table 2), place at 100°C for 5 minutes, then place at room temperature, gradually cool, denature and anneal to form a band Fragments with cohesive ends (ie, g++/g--primer annealing products).

The SK-gRNA was digested with AarI (the enzyme and the reagents used in the system were all purchased from Ferment Company) to form a vector with cohesive ends. The enzyme digestion reaction system was as follows:

Remarks: SK-gRNA can come from application number 201510485573.2.

Digestion at 37°C for 3 hours, followed by purification with the Biomed Gel Recovery Kit (Biomed, DR0103) according to the product instructions; the linear vector SK-gRNA/AarI was obtained.

The carrier and the fragment were connected with T4 enzyme (purchased from NEB Company), and the reaction was as follows:

React at room temperature for 1 hour. 5 μL of the ligation product was transformed into Escherichia coli competent cell DH5α to obtain the ligation plasmid SK-gRNA ^GL1-1 . Using primer T7: 5'-TAATACGACTCACTATAGG-3', sequencing confirmed that the clone was constructed correctly. That is, VRER and target site-related sequences are contained.

The correctly sequenced SK-gRNA ^GL1-1 plasmid was double-digested with KpnI and BglII, and purified with the Biomed Gel Extraction Kit (Biomed, DR0103) according to the product instructions to obtain a linear fragment. This linear fragment was connected between the BamHI and KpnI recognition sites of the pC1300-VRER binary vector (i.e., the recombinant plasmid pC1300-VRER obtained in Example 1) to obtain the final knockout rice binary expression vector pC1300-VRER-gRNA ^{GL1 -1} .

KpnI, BglII, BamHI and other reagents in the system were purchased from TAKARA Company, and the double enzyme digestion system was as follows:

Enzyme digestion at 37°C for 3 hours, followed by purification with the Biomed Gel Extraction Kit (Biomed, DR0103) according to the product instructions; the linear fragment gRNA ^GL1-1 /KpnI+BglII was obtained.

Digestion at 37°C for 3 hours, followed by purification with the Biomed Gel Recovery Kit (Biomed, DR0103) according to the product instructions; the linear vector pC1300-VRER/KpnI+BamHI was obtained.

The ligation reaction is as follows:

React at room temperature for 1 hour. Take 5 μL of the ligation product and use it to transform Escherichia coli competent cell DH5 to obtain the ligation plasmid. Using primer pC1300-F: 5'-ACACTTTATGCTTCCGGCTC-3', sequencing confirmed that the clone was constructed correctly. When the sequencing result matches the design sequence (including the target site sequence), it is determined that the construction is correct; otherwise, the construction is incorrect.

Table 2 GL1-1 target sites, oligonucleotides, primers

Embodiment 3, the acquisition of transgenic plants

The above-mentioned final knockout rice binary expression vector pC1300-VRER-gRNA ^GL1-1 was transformed into Agrobacterium strain EHA105 by electric shock method, and the binary expression vector was transformed into rice Nipponbare by Agrobacterium-mediated method mature embryonic callus. The specific method of transformation is to cut out mature embryos of Nipponbare seeds after sterilization, and inoculate them into the culture medium for inducing callus. After culturing for 1 week, vigorously growing, pale yellow, relatively loose embryogenic calli were selected to be used as recipients for transformation. Rice calli were infected with the EHA105 strain containing the pC1300-VRER-gRNA ^GL1-1 plasmid, and after culturing for 3 days at 25°C in the dark, the resistant calli and transgenic plants. The transgenic plants growing normally on the hygromycin selection medium were selected for identification.

Embodiment 4, identification of transgenic plants

1. Enzyme digestion identification

Use molecular biology methods to identify the mutation of the target gene. The wild-type control and the transgenic plant genomic DNA obtained in Example 3 were extracted from a single plant by the CTAB method, and the target band was amplified by PCR using the primers described in Table 2 and KODFX DNA polymerase (purchased from Toyobo Biotechnology Co., Ltd.). The reaction conditions were as follows:

Reaction program: denaturation at 94°C for 2 minutes; then denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 68°C for 40 seconds, and 34 cycles of amplification; finally extension at 68°C for 5 minutes.

The obtained PCR product, about 688bp, was detected by enzyme digestion using NcoI (purchased from NEB Company), and the system was as follows:

37 DEG C enzyme digestion 3 hours, 2% agarose gel electrophoresis (145V) 25min, the result is as shown in Figure 2, is completely degraded into the sample of two smaller bands of 428bp and 260bp for wild type and knockout negative ( Such as wt and 2～4,8～10,13,18～20); samples that are not degraded at all are knockout homozygotes (such as 14); samples containing two band types are knockout heterozygotes (such as 1, 5,6,7,11,12,15,16,17).

2. Sequencing

The above PCR products were sent to a sequencing company (Hangzhou Qingke Zixi Biotechnology Co., Ltd.), and sequenced using F in Table 2 as a primer. The obtained results were compared with the wild-type sequence. If the sequencing result is bimodal, use the degenerate codon strategy analysis (http://dsdecode.scgene.com/ for peak map analysis), and the heterozygous mutation information can be obtained directly. The results are shown in Figure 3, and knockout homozygotes and heterozygotes were detected to varying degrees.

3. Phenotyping

As shown in Figure 4, when water is dripped onto the surface of the leaf, if the water can maintain the shape of the water drop, it can be explained that the rice leaf surface has wax synthesis, which is the wild type. On the contrary (that is, the water drop shape cannot be maintained), it indicates that the waxy gene has been destroyed, and it is a knockout positive plant.

in conclusion:

The Cas9 protein expressed by the original Cas9 gene can only realize the genome editing function when the PAM region is 5'-NGG-3'. Compared with the original Cas9 protein, VRER (variant Cas9 protein) can realize genome editing function when the PAM region is 5'-NGCG-3', while ordinary Cas9 protein cannot realize editing in this case. Remarks: N represents any nucleotide of A, T, C, and G. In this case (Figure 3), N is G.

Example 5. Change the gene GL1-1 in Examples 2 to 4 to LG1 (including GL1-1 contained in the superscript, all changed to LG1), and correspondingly, change "5'-ACGTTGCAGTGGC CCATGG CGCG" to "5'-GCCAAGAAGAG CTGCAG GAAGCG", "CGCG" was changed to "AGCG", "NcoI" was changed to "PstI"; the system and method used were unchanged, and the oligonucleotide primers used g++/g- - becomes "g++: GGCAGCCAAGAAGAGCTGCAGGA; g--: AAACTCCTGCAGCTCTTCTTGGC"; primer F/R becomes "F: GATCTGGCCTGAGCAACCT; R: CCCCAGTAGCTGTGTCTCG".

The results are as follows: Genomic DNA of the wild-type control and the obtained transgenic plants was extracted from a single plant by the CTAB method, and the target band was amplified by PCR using the above primers and KODFX DNA polymerase (purchased from Toyobo Biotechnology Co., Ltd.). The reaction conditions were as follows:

Reaction program: denaturation at 94°C for 2 minutes; then denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 68°C for 40 seconds, and 34 cycles of amplification; finally extension at 68°C for 5 minutes.

The PCR product, about 671bp, was sequenced with the modified F as the primer. The obtained results were compared with the wild-type sequence. If the sequencing result is bimodal, use the degenerate codon strategy analysis (http://dsdecode.scgene.com/ for peak map analysis), and the heterozygous mutation information can be obtained directly. The results are shown in Figure 5, with varying degrees of mutation detection.

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims (6)

1. Plant Cas9 variant protein VRER, characterized in that: the amino acid sequence of the protein is shown in SEQ ID No: 2. 2. Plant Cas9 variant protein VRER, characterized in that: the protein also includes derivatives generated by adding, substituting, inserting and deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO:2. 3. The plant Cas9 variant gene VRER, characterized in that: the gene encodes the plant Cas9 variant protein VRER, and the nucleotide sequence of said gene is shown in SEQ ID NO:1. 4. The plant Cas9 variant gene VRER is characterized in that: the gene also includes mutants generated by adding, substituting, inserting and deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 1, etc. genes and derivatives. 5. The recombinant expression vector is characterized in that it comprises the gene according to claim 3 or 4. 6. The use of plant Cas9 variant protein VRER as claimed in claim 1 or 2, characterized in that: the genome editing function can be realized when the PAM region is 5'-NGCG-3'.