CN114438056A - CasF2 protein, CRISPR/Cas gene editing system and its application in plant gene editing - Google Patents

Abstract

The invention discloses a CasF2 protein, a CRISPR/Cas gene editing system and application thereof in plant gene editing, relating to the technical field of gene editing. The amino acid sequence of the CasF2 protein is shown as SEQ ID No. 2; or has substitution, deletion or addition of one or more amino acids compared with the sequence shown in SEQ ID No.2 and has the same or similar biological functions. The CRISPR/Cas gene editing system comprises a CasF2 protein or a plasmid expressing the CasF2 protein. The invention provides a novel efficient and stable gene editing system, which can use shorter sgRNA to guide CasF2 to edit a target sequence to generate insertion or deletion of a DNA sequence.

Description

CasF2 protein, CRISPR/Cas gene editing system and its application in plant gene editing

technical field

The invention relates to the technical field of gene editing, in particular to a CasF2 protein, a CRISPR/Cas gene editing system and its application in plant gene editing.

Background technique

Gene editing technology is an emerging scientific technology in recent years, which can be used for targeted silencing and modification of genes to create new non-transgenic materials. CRISPR/Cas (clustered regularly interspaced shortpalindromic repeats and CRISPR-associated protein) system is simple to construct, low cost and high precision, and can achieve multiple gene editing at the same time with high efficiency. It is a gene targeting modification with good application prospects. technology.

At present, Cas9, Cas12 and Cas13 are mainly in the CRISPR/Cas system. However, the molecular weights of these Cas genes are large, and the range of PAM recognition sequences is very narrow. Therefore, it is urgent to develop new Cas genes for gene editing in plants. The development of new gene editing systems can provide effective tools for crop gene editing breeding to achieve the desired goals.

In the Cas gene family, there is no detailed report on the use of CasF2 gene to construct CRISPR gene editing vector for application in crops such as Arabidopsis, tobacco, soybean and maize.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide CasF2 protein, CRISPR/Cas gene editing system and its application in plant gene editing; the present invention provides a new efficient and stable gene editing system, which can use shorter sgRNA to guide CasF2 to perform Editing of target sequences, resulting in insertions or deletions of DNA sequences.

The technical scheme provided by the present invention is as follows:

In one aspect, the present invention provides a CasF2 protein, the amino acid sequence of the CasF2 protein is shown in SEQ ID No. 2; or the amino acid sequence of the CasF2 protein is compared with the sequence shown in SEQ ID NO. Substitution, deletion or addition of multiple amino acids with the same or similar biological function.

The amino acid sequence shown in SEQ ID No.2 is as follows:

MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAPKPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKSEEEPPNFQPPAKCHVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGKSSESHAAWFAATGVSNHGYSHVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEEEEVATNETGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYAGYVRDPNAPIPLGVVRNRCDIQKGCPGYIPEWQREAGTAISPKTGKAVTVPGLSPKKNKRMRRYWRSEKEKAQDALLVTVRIGTDWVVIDVRGLLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTLDACGTYARKWTLKGKQTKATLDKLTATQTVALVAIDLGQTNPISAGISRVTQENGALQCEPLDRFTLPDDLLKDISAYRIAWDRNEEELRARSVEALPEAQQAEVRALDGVSKETARTQLCADFGLDPKRLPWDKMSSNTTFISEALLSNSVSRDQVFFTPAPKKGAKKKAPVEVMRKDRTWARAYKPRLSVEAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSINYVIEKTRRRTQCQIVIPVIEDLNVRFFHGSGKRLPGWDNFFTAKKENRWFIQGLHKAFSDLRTHRSFYVFEVRPERTSITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTGKTMPKREEPRDAQGTAPARKTKKASKSKAPPAEREDQTPAQEPSQTSKRPAATKKAGQAKKKK。

The CasF2 protein is an RNA nuclease, and has the following activities: the activity of binding to a specific site of the target sequence and cleaving nucleic acid, or the activity of recognizing the PAM site and the endonuclease activity. In one embodiment, the amino acid sequence of the CasF2 protein can be compared with the amino acid sequence shown in SEQ ID No. 2, and its sequence similarity is more than 95%, preferably more than 97%, more preferably more than 98% , most preferably 99% or more, as long as it has the same function as the amino acid sequence shown in SEQ ID No. 2.

The present invention also provides the encoding gene of the CasF2 protein, and the nucleotide sequence of the encoding gene is shown in the following SEQ ID No.1:

ATGGATTACAAGGATCACGACGGTGATTACAAGGACCATGATATCGACTACAAGGATGACGATGACAAGATGGCGCCTAAGAAGAAGAGAAAAGTGGGAATTCACGGTGTTCCTGCTGCCCCAAAGCCGGCCGTGGAGTCAGAGTTCTCTAAGGTTCTTAAGAAGCATTTCCCGGGCGAGCGCTTCAGATCTTCCTACATGAAGAGGGGCGGAAAGATCCTGGCGGCTCAGGGAGAGGAGGCTGTGGTTGCCTACCTCCAGGGCAAGAGCGAGGAGGAGCCTCCAAACTTCCAGCCGCCTGCTAAGTGCCACGTCGTGACTAAGTCAAGAGATTTCGCCGAGTGGCCAATTATGAAGGCGTCAGAGGCTATCCAGCGCTACATTTACGCTCTGTCTACTACCGAGAGAGCCGCGTGCAAGCCGGGCAAGTCTTCTGAGAGCCACGCTGCTTGGTTCGCTGCTACAGGCGTCTCAAACCACGGATACTCTCATGTGCAGGGACTCAATCTTATCTTCGATCATACTCTCGGAAGATACGACGGCGTTCTGAAGAAGGTCCAGCTCCGCAACGAGAAGGCTAGGGCCCGCCTTGAGTCAATCAATGCTTCTAGGGCTGACGAGGGCCTGCCTGAGATTAAGGCTGAGGAGGAGGAGGTCGCCACCAACGAGACAGGTCATCTCCTTCAGCCACCGGGCATCAATCCATCCTTCTACGTGTACCAGACAATTAGCCCTCAGGCTTACAGACCAAGGGATGAGATCGTGCTCCCTCCAGAGTACGCGGGTTACGTTAGAGATCCTAACGCTCCTATTCCACTTGGCGTTGTCCGCAATAGATGCGATATCCAGAAGGGCTGCCCAGGATACATTCCGGAGTGGCAGAGGGAGGCTGGAACTGCCATTTCCCCGAAGACTGGAAAGGCCGTCACCGTGCCTGGCCTCAGCCCAAAGAAGAACAAGAGGATGAGAAGGTACTGGCGCTCAGAGAAGGAGAAGGCGC AGGACGCTCTGCTCGTTACAGTCAGAATCGGAACTGATTGGGTGGTTATTGACGTGCGCGGTCTTCTGAGAAACGCGAGGTGGCGCACTATCGCTCCAAAGGATATTTCTCTTAATGCCCTCCTTGACCTGTTCACCGGAGATCCGGTCATCGACGTGCGCAGAAATATTGTTACCTTCACATACACTCTCGATGCTTGCGGCACCTACGCCAGGAAGTGGACACTTAAGGGAAAGCAGACTAAGGCTACCCTTGATAAGCTGACCGCCACACAGACTGTTGCCCTTGTCGCGATCGACCTGGGCCAGACCAACCCTATCTCCGCCGGAATTAGCCGCGTCACACAGGAGAATGGTGCGCTCCAGTGCGAGCCACTTGATAGATTCACTCTGCCGGATGACCTGCTCAAGGATATCTCTGCGTACAGAATTGCTTGGGACAGGAACGAGGAGGAGCTTAGAGCGAGGTCCGTTGAGGCTCTGCCAGAGGCTCAGCAGGCCGAAGTTAGGGCTCTCGATGGCGTCAGCAAGGAGACAGCCAGGACTCAGCTCTGCGCGGATTTCGGACTCGACCCGAAGAGGCTTCCTTGGGATAAGATGTCTTCCAACACAACTTTCATCTCAGAGGCTCTTCTGAGCAATTCAGTCTCTCGCGACCAGGTGTTCTTCACCCCGGCCCCTAAGAAGGGTGCTAAGAAGAAGGCGCCTGTGGAGGTTATGCGCAAGGATAGAACATGGGCGCGCGCTTACAAGCCAAGACTCTCTGTGGAGGCCCAGAAGCTTAAGAATGAGGCGCTCTGGGCTCTTAAGAGAACCTCCCCAGAGTACCTGAAGCTCAGCAGGCGCAAGGAGGAGCTTTGCAGAAGGTCTATCAACTACGTTATTGAGAAGACACGCAGAAGGACTCAGTGCCAGATCGTGATTCCGGTTATTGAGGATCTTAATGTCAGATTCTTCCACGGTTCCGGCAAGAGGCTGCCTGGTTGGGACAACTTCTTCAC TGCCAAGAAGGAGAATAGATGGTTCATCCAGGGCCTGCACAAGGCGTTCTCAGATCTCAGGACCCATCGCTCTTTCTACGTCTTCGAAGTGAGGCCTGAGAGGACCTCTATTACATGCCCAAAGTGCGGACACTGCGAGGTTGGAAACAGGGACGGAGAGGCTTTCCAGTGCCTCTCCTGCGGAAAGACCTGCAACGCCGATCTTGACGTTGCGACCCATAATCTGACACAGGTCGCCCTCACTGGAAAGACCATGCCGAAGAGGGAGGAGCCTAGGGATGCTCAGGGTACTGCTCCAGCTAGGAAGACCAAGAAGGCTTCCAAGAGCAAGGCTCCACCTGCTGAGAGGGAGGACCAGACACCAGCTCAGGAGCCGTCCCAGACAAGCAAGAGGCCTGCCGCGACTAAGAAGGCTGGACAGGCTAAGAAGAAGAAGTGA。

The CasF2 gene is a Cas12j-type Cas gene with the potential to delete long DNA sequences. The PAM recognition sequence of CasF2 gene is: 5'-TBN-3' (B=G/T/C), and CasF2 is highly specific to the recognized target sequence site. The target sequence of the CasF2 editing system is 18 bp. CasF2 has more editable sites on genomic DNA and has a wider range of potential targets.

The present invention may also include a nucleotide sequence that is reverse complementary to the nucleotide sequence of the CasF2 protein shown in SEQ ID No. 1.

In another aspect, the present invention also provides an expression vector comprising the aforementioned nucleotide sequence. The expression vector can express the CasF2 protein in the target cells, so that the corresponding gene editing can be performed in the target cells. Commonly used vectors may be, for example, the pBI121 vector, the PCAMBIA1300 vector, and the like.

The present invention may also include recombinant cells containing the expression vector, preferably, the recombinant cells are eukaryotic cells, more preferably, the recombinant cells are plant cells.

In another aspect, the present invention also provides the application of the CasF2 protein, the gene encoding the CasF2 protein or the expression vector in gene editing.

In another aspect, the present invention also provides a CRISPR/Cas gene editing system, the gene editing system comprising the aforementioned CasF2 protein or a plasmid expressing the CasF2 protein.

In one embodiment, the gene editing system further comprises an sgRNA or a plasmid expressing the sgRNA, eg, the expression plasmid contains the gene encoding the sgRNA.

The above two sgRNAs can be used to guide the CasF2 protein to perform gene editing at the target gene sequence position in the receptor material. When the sgRNA3 sequence shown in SEQ ID No. 3 is used, the CasF2 protein has the function of sgRNA sequence processing, and the sgRNA3 Cut into a dgRNA1 sequence to guide CasF2 for gene editing at the target site. The sgRNA can be one or more sgRNAs for targeting one or more target genes. The sgRNA includes nucleotides containing its corresponding recognition region sequence.

In another aspect, the present invention provides the application of the CRISPR/Cas gene editing system in plant gene editing; preferably, the plant is a monocotyledonous plant or a dicotyledonous plant; more preferably, the plant is selected from the group consisting of Any of Arabidopsis, tobacco, soybean, and corn.

In one embodiment, the application comprises the steps of:

(a) determining the sgRNA of the gene to be edited according to the PAM recognition site of the CasF2 protein;

(b) cloning the gene sequence of the CasF2 protein and the corresponding sgRNA into an expression vector;

(c) transforming the expression vector into Agrobacterium, and introducing the expression vector into a target plant through an Agrobacterium-mediated genetic transformation method to obtain a targeted gene editing plant;

Preferably, in the genetic transformation, _MgCl2 is used to activate gene editing activity.

The editing activity of the CasF2 gene requires a certain concentration of Mg ²⁺ to activate. When CasF2 exists in plants, without adding a certain concentration of Mg ²⁺ , CasF2 does not edit the target site in cells.

In one embodiment, the plant is Arabidopsis thaliana, and the nucleotide sequence (sgRNA3 sequence) corresponding to the sgRNA is shown in SEQ ID No.3. Specifically, SEQ ID No. 3:

TAATGTCGGAACGCTCAACGATTGCCCCCTCACGAGGGGAC-N ₁₈ (target sequence).

In one embodiment, the plant is tobacco, and the nucleotide sequence (sgRNA1 sequence) corresponding to the sgRNA is as shown in SEQ ID No.4. Specifically, SEQ ID No. 4:

CGCTCAACGATTGCCCCCTCACGAGGGGAC-N ₁₈ (target sequence).

In one embodiment, the present invention provides the sgRNA target sequence of plant Arabidopsis thaliana BRI1 gene; the nucleotide sequence of the sgRNA target sequence is shown in SEQ ID No.5.

Specifically, SEQ ID No. 5: CTGCGAATTCAATCTCCG.

In one embodiment, the present invention provides the sgRNA target sequence of the plant tobacco PDS gene; the nucleotide sequence of the sgRNA target sequence is shown in SEQ ID No.6.

Specifically, SEQ ID No. 6: GTAGTAGCGACTCCATGG.

Based on the application of the CRISPR/Cas gene editing system of the present invention in plant gene editing, the present invention also provides a method for gene editing of plants using the gene editing vector system of the present invention.

In the present invention, the CasF2 protein and sgRNA in the CRISPR/Cas system can be in the same expression vector or in different expression vectors.

In the present invention, the abbreviation "CRISPR" or "crispr" both refer to the acronym of Clustered regularly interspaced short palindromic repeats, namely Clustered regularly interspaced short palindromic repeats. In a nucleotide sequence, when expressing a base, unless otherwise specified, the base represented by the letter N has the usual meaning in the art, that is, N represents random or any base A, T, C or G.

As used herein, "comprising," "having," or "including" includes "comprising," "consisting essentially of," "consisting essentially of," and "consisting of."

Beneficial effects:

The present invention provides a novel CasF2 protein and an efficient and stable Cas-mediated gene editing vector, which can be stably and efficiently expressed in plant cells, thereby better regulating the expression of target genes. CasF2 protein has a small number of amino acids, the small size of CasF2 provides advantages for delivery into plant cells (easy packaging and delivery), and CasF2 uses a single active site for crRNA processing and cleavage of DNA sequences. CasF2 has a wide range of PAM recognition sites, and can construct multiple gene editing site vectors to achieve knockout of more genes. The present invention can be used for gene editing of DNA sequences in Arabidopsis, tobacco, soybean and maize.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the accompanying drawings required in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is the PCR sequencing result provided in the embodiment of the present invention;

2 is a phenotypic diagram of an Arabidopsis BRI1 gene-edited plant obtained by the CRISPR/CasF2 gene-editing system according to an embodiment of the present invention;

Fig. 3 is the schematic diagram of the sgRNA3 T plasmid vector provided in the embodiment of the present invention;

Fig. 4 is the schematic diagram of pHCasF2 carrier provided in the embodiment of the present invention;

Fig. 5 is the schematic diagram of the sgRNA1T plasmid provided in the embodiment of the present invention;

Fig. 6 is the tobacco tissue culture result graph that the embodiment of the present invention provides;

FIG. 7 is a sequence diagram of the tobacco PDS target sequence provided by the embodiment of the present invention.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

1. Targeting strategy of Arabidopsis target gene BRI1:

Using the brassinosteroid receptor BRI1 of Arabidopsis thaliana as the target gene, CRISPR/CasF2 was used to directionally edit Arabidopsis BRI1.

CasF2/sgRNA design:

According to the PAM sequence recognized by CasF2, the nucleotide sequence of the sgRNA target of Arabidopsis BRI1 was selected (target sequence SEQ ID No. 5) (5'-3'): CTGCGAATTCAATCTCCG.

2. Editing of the Arabidopsis BRI1 gene

The sgRNA of the Arabidopsis BRI1 gene was constructed, and the Arabidopsis AtU6-26 promoter was selected to drive the expression of the sgRNA sequence; at the same time, the AtU6-26 terminator sequence was added after the sgRNA sequence. CRISPR/CasF2 gene editing vector driven by sgRNA driven by Arabidopsis U6 promoter.

KOD-plus neo was used to amplify the targeted sgRNA sequence with the sgRNA3 T plasmid (Fig. 3 is a schematic diagram of the plasmid vector) as a template. The first round of PCR, amplification system (20μL):

Primer pair: U6-26p-F+F2BRI1-gRNA-R to amplify 1 tube, primer pair: F2BRI1-U6-26t-F+U6-26t-R to amplify 1 tube.

U6-26p-F (SEQ ID No. 7):

TTCAGAggtctcTtagtCGACTTGCCTTCCGCACAATAC;

U6-26t-R (SEQ ID No. 8):

AGCGTGggtctcGcgccTATTGGTTTATCTCATCGGAAC;

F2BRI1-U6-26t-F (SEQ ID No. 9):

(The bold underlined part is the target sequence)

F2BRI1-gRNA-R (SEQ ID No. 10):

CGGAGATTGAATTCGCAGGTCCCCTCGTGAGGGGCAATC.

PCR reaction program:

After PCR amplification, 2 μL of PCR products in 2 tubes were taken as templates for the second round of PCR.

Amplification system:

Reaction program:

After the second round of PCR, take 1 μL of the PCR product as the template for the third round of PCR. The reaction system and amplification system were the same as in the first round. After amplification, agarose gel electrophoresis was performed.

After the PCR product was recovered by gel, it was ligated with the pHCasF2 vector (Fig. 4 is a schematic diagram of the vector).

Connection system:

Ligation reaction: 37°C for 5 min, 10°C for 5 min; 20°C for 5 min; 15 cycles, and finally 37°C for 5 min.

The ligation product was transformed into E. coli DH5α competent, and the competent was spread on LB (Kan ⁺ ) plates. Pick colonies from the plate and send them to the company for sequencing verification.

The constructed BRI1 gene targeting expression vector pHCasF2-BRI1 was transformed into Agrobacterium GV3101.

Agrobacterium was picked to infect Arabidopsis thaliana in full bloom. The steps of infecting Arabidopsis thaliana by Agrobacterium method are as follows: 20 μL of Agrobacterium strain is drawn and added to 3 mL of yeast liquid medium (Yeast Extract Broth, YEB) (kan ⁺ , Rif ⁺ ); 28°C, 200rpm, shake for 24h;

Take the shaken bacteria, centrifuge at 12,000 rpm for 1 min to collect the strains, remove the supernatant, resuspend the pellet with 1 mL of 5% sucrose 1/2 MS solution, and collect the strains again; finally resuspend with 1 mL of 5% sucrose 1/2 MS solution, while Add 0.2 μL of Silwet77 and a final concentration of 10 mM MgCl ₂ , and after thorough mixing, Arabidopsis can be infected.

Screen and sequence resistant shoots:

After the Arabidopsis seeds are mature, the seeds are harvested. Seeds were sown on 1/2 MS medium plates containing 50 mg/L hygromycin, and resistant shoots were picked.

PCR amplifies the DNA sequence near the target site, and the PCR product is sent to the company for sequencing. Photographs of Arabidopsis plants with the BRI1 gene edited.

PCR amplification primers for DNA amplification near the target sequence:

F2BRI1crispr-identify-F (SEQ ID No. 11):

CTGGCTTCAAGTGCTCTGCTTC;

F2BRI1crispr-identify-R (SEQ ID No. 12):

GTAATTCGCCGGAAAACTCG.

The amplification system was the same as the first round PCR.

Analysis of results:

(1) No BRI1 gene-edited plants were found in the infection solution of Arabidopsis thaliana without adding a final concentration of 10 mM MgCl ₂ , but only Arabidopsis plants infected with the infection solution with 10 mM MgCl ₂ added, Only plants with the BRI1 gene edited have emerged.

(2) Amplify the DNA sequence near the BRI1 target. It is found by sequencing that the target has a set of peaks (shown in Figure 1); and the Arabidopsis plant has a sterile phenotype, which is consistent with the reported literature results ( shown in Figure 2).

The results showed that the gene editing of the CasF2 protein in Arabidopsis requires the presence of MgCl ₂ , and the gene editing of the Arabidopsis thaliana gene can produce insertion and deletion of DNA sequences.

Example 2

1. N. benthamiana PDS gene targeting strategy:

The phytoene dehydrogenase PDS of Nicotiana benthamiana was used as the target gene, and the PDS of Nicotiana benthamiana was edited using CRISPR/CasF2.

CasF2/sgRNA design:

According to the PAM sequence recognized by CasF2, the nucleotide sequence of the sgRNA target of N. benthamiana PDS was selected (target sequence SEQ ID No. 6) (5'-3'): GTAGTAGCGACTCCATGG.

2. Editing of the Arabidopsis BRI1 gene

Editing of N. benthamiana PDS gene

To construct the sgRNA of the tobacco PDS gene, the construction method is the same as that of Arabidopsis. The Arabidopsis thaliana BRI1 target primer is replaced with the tobacco PDS target primer, and the template for amplifying the sgRNA sequence is replaced by the sgRNA1T plasmid (Figure 5 is a schematic diagram of the vector). The target primers for the tobacco PDS gene are as follows:

F2NtPDS-U6-26t-F (SEQ ID No. 13):

(The bold underlined part is the target sequence)

F2NtPDS-gRNA-R (SEQ ID No. 14):

CCATGGAGTCGCTACTACGTCCCCTCGTGAGGGGCAATC.

The constructed tobacco targeting PDS gene plasmid pHCasF2-PDS was transformed into Agrobacterium GV3101, and the leaf disc method was used to transform tobacco. The specific steps were as follows:

1. Leaf disinfection:

1) Select the leaves of Nicotiana benthamiana that grow from 1-2 months into a beaker, soak in sterile water for 20-30min;

2) Discard sterile water, soak the leaves with 0.6% sodium hypochlorite (add 1 drop of Tween-20 to every 50 mL of solution), and let the leaves soak in water for 10 minutes;

3) Rinse 5 times with sterile water,

2. Leaf infection:

1) Cut off the edge of the leaf with sterilized scissors or a hole punch, cut the leaf into 1cm ² size, avoid the main vein of the leaf, and put the leaf into the liquid co-cultivation medium;

2) transfer all the cut leaves into the infection solution, infect 8min; then use sterile filter paper to absorb the infection solution on the leaf surface;

3) Put the leaves on the solid co-medium quickly, with the back of the leaves facing down, and cultivate in the dark at 25°C for 48h;

4) The leaves were transferred to the selection medium, and cultured at 25°C under 16h light/8h dark, and the selection medium was replaced every 10 days until the transformed cells differentiated into adventitious buds.

5) Select the albino seedlings for photography, extract the genomic DNA of the albino seedlings, use primers to amplify the DNA sequence near the target, and send the PCR product to the company for sequencing.

PCR amplification primers for DNA amplification near the target sequence:

NtPDS-seq-F (SEQ ID No. 15): ATGCCCCAAATTGGACTTGTTTC;

NtPDS1-23-R (SEQ ID No. 16): CTGGAGCGTGGACAACATAAAT.

The method for constructing tobacco sgRNA targets is the same as that for constructing the Arabidopsis system.

Analysis of results:

According to the gene editing results of Arabidopsis thaliana, the CasF2 protein needs the presence of MgCl ₂ for gene editing to be active, and the target DNA sequence is edited under the guidance of sgRNA. Therefore, in tobacco PDS gene tissue culture, the final concentration of 100nM MgCl ₂ was added to the screening medium; after Agrobacterium infection, after 2 rounds of screening medium, albino seedlings were produced (see Figure 6). In selection medium without the addition of _MgCl2 , green differentiated shoots were produced. The target sequence of the albino seedlings was sequenced, and it was found that there was a base deletion in the target sequence. Figure 7. Tobacco PDS target sequence sequencing.

Meanwhile, the sgRNA3 sequence was used as the sgRNA sequence in Arabidopsis, and the sgRNA1 sequence was used as the sgRNA sequence in tobacco. Through the analysis of the constructed Arabidopsis sgRNA3 guide sequence and tobacco sgRNA1 guide sequence, it was found that the CasF2 protein can process pre-crRNA, and the sgRNA1 with a shorter sequence can be used to guide CasF2 to cut the target sequence. In this way, multiple sgRNAs can be constructed simultaneously for multi-gene targeting.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

SEQUENCE LISTING

<110> Jilin Academy of Agricultural Sciences

<120> CasF2 protein, CRISPR/Cas gene editing system and its application in plant gene editing

<130> PA20037888

<160> 16

<170> PatentIn version 3.3

<210> 1

<211> 2439

<212> DNA

<213> CasF2 gene

<400> 1

atggattaca aggatcacga cggtgattac aaggaccatg atatcgacta caaggatgac 60

gatgacaaga tggcgcctaa gaagaagaga aaagtgggaa ttcacggtgt tcctgctgcc 120

ccaaagccgg ccgtggagtc agagttctct aaggttctta agaagcattt cccgggcgag 180

cgcttcagat cttcctacat gaagaggggc ggaaagatcc tggcggctca gggagaggag 240

gctgtggttg cctacctcca gggcaagagc gaggaggagc ctccaaactt ccagccgcct 300

gctaagtgcc acgtcgtgac taagtcaaga gatttcgccg agtggccaat tatgaaggcg 360

tcagaggcta tccagcgcta catttacgct ctgtctacta ccgagagagc cgcgtgcaag 420

ccgggcaagt cttctgagag ccacgctgct tggttcgctg ctacaggcgt ctcaaaccac 480

ggatactctc atgtgcaggg actcaatctt atcttcgatc atactctcgg aagatacgac 540

ggcgttctga agaaggtcca gctccgcaac gagaaggcta gggcccgcct tgagtcaatc 600

aatgcttcta gggctgacga gggcctgcct gagattaagg ctgaggagga ggaggtcgcc 660

accaacgaga caggtcatct ccttcagcca ccgggcatca atccatcctt ctacgtgtac 720

cagacaatta gccctcaggc ttacagacca agggatgaga tcgtgctccc tccagagtac 780

gcgggttacg ttagagatcc taacgctcct attccacttg gcgttgtccg caatagatgc 840

gatatccaga agggctgccc aggatacatt ccggagtggc agagggaggc tggaactgcc 900

atttccccga agactggaaa ggccgtcacc gtgcctggcc tcagcccaaa gaagaacaag 960

aggatgagaa ggtactggcg ctcagagaag gagaaggcgc aggacgctct gctcgttaca 1020

gtcagaatcg gaactgattg ggtggttatt gacgtgcgcg gtcttctgag aaacgcgagg 1080

tggcgcacta tcgctccaaa ggatatttct cttaatgccc tccttgacct gttcaccgga 1140

gatccggtca tcgacgtgcg cagaaatatt gttaccttca catacactct cgatgcttgc 1200

ggcacctacg ccaggaagtg gacacttaag ggaaagcaga ctaaggctac ccttgataag 1260

ctgaccgcca cacagactgt tgcccttgtc gcgatcgacc tgggccagac caaccctatc 1320

tccgccggaa ttagccgcgt cacacaggag aatggtgcgc tccagtgcga gccacttgat 1380

agattcactc tgccggatga cctgctcaag gatatctctg cgtacagaat tgcttgggac 1440

aggaacgagg aggagcttag agcgaggtcc gttgaggctc tgccagaggc tcagcaggcc 1500

gaagttaggg ctctcgatgg cgtcagcaag gagacagcca ggactcagct ctgcgcggat 1560

ttcggactcg acccgaagag gcttccttgg gataagatgt cttccaacac aactttcatc 1620

tcagaggctc ttctgagcaa ttcagtctct cgcgaccagg tgttcttcac cccggcccct 1680

aagaagggtg ctaagaagaa ggcgcctgtg gaggttatgc gcaaggatag aacatgggcg 1740

cgcgcttaca agccaagact ctctgtggag gcccagaagc ttaagaatga ggcgctctgg 1800

gctcttaaga gaacctcccc agagtacctg aagctcagca ggcgcaagga ggagctttgc 1860

agaaggtcta tcaactacgt tattgagaag acacgcagaa ggactcagtg ccagatcgtg 1920

attccggtta ttgaggatct taatgtcaga ttcttccacg gttccggcaa gaggctgcct 1980

ggttgggaca acttcttcac tgccaagaag gagaatagat ggttcatcca gggcctgcac 2040

aaggcgttct cagatctcag gacccatcgc tctttctacg tcttcgaagt gaggcctgag 2100

aggacctcta ttacatgccc aaagtgcgga cactgcgagg ttggaaacag ggacggagag 2160

gctttccagt gcctctcctg cggaaagacc tgcaacgccg atcttgacgt tgcgacccat 2220

aatctgacac aggtcgccct cactggaaag accatgccga agagggagga gcctagggat 2280

gctcagggta ctgctccagc taggaagacc aagaaggctt ccaagagcaa ggctccacct 2340

gctgagaggg aggaccagac accagctcag gagccgtccc agacaagcaa gaggcctgcc 2400

gcgactaaga aggctggaca ggctaagaag aagaagtga 2439

<210> 2

<211> 812

<212> PRT

<213> Amino acid sequence of CasF2 protein

<400> 2

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

1 5 10 15

Tyr Lys Asp Asp Asp Asp Lys Met Ala Pro Lys Lys Lys Arg Lys Val

20 25 30

Gly Ile His Gly Val Pro Ala Ala Pro Lys Pro Ala Val Glu Ser Glu

35 40 45

Phe Ser Lys Val Leu Lys Lys His Phe Pro Gly Glu Arg Phe Arg Ser

50 55 60

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

65 70 75 80

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

85 90 95

Phe Gln Pro Pro Ala Lys Cys His Val Val Thr Lys Ser Arg Asp Phe

100 105 110

Ala Glu Trp Pro Ile Met Lys Ala Ser Glu Ala Ile Gln Arg Tyr Ile

115 120 125

Tyr Ala Leu Ser Thr Thr Glu Arg Ala Ala Cys Lys Pro Gly Lys Ser

130 135 140

Ser Glu Ser His Ala Ala Trp Phe Ala Ala Thr Gly Val Ser Asn His

145 150 155 160

Gly Tyr Ser His Val Gln Gly Leu Asn Leu Ile Phe Asp His Thr Leu

165 170 175

Gly Arg Tyr Asp Gly Val Leu Lys Lys Val Gln Leu Arg Asn Glu Lys

180 185 190

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

195 200 205

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

210 215 220

Gly His Leu Leu Gln Pro Pro Gly Ile Asn Pro Ser Phe Tyr Val Tyr

225 230 235 240

Gln Thr Ile Ser Pro Gln Ala Tyr Arg Pro Arg Asp Glu Ile Val Leu

245 250 255

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

260 265 270

Leu Gly Val Val Arg Asn Arg Cys Asp Ile Gln Lys Gly Cys Pro Gly

275 280 285

Tyr Ile Pro Glu Trp Gln Arg Glu Ala Gly Thr Ala Ile Ser Pro Lys

290 295 300

Thr Gly Lys Ala Val Thr Val Pro Gly Leu Ser Pro Lys Lys Asn Lys

305 310 315 320

Arg Met Arg Arg Tyr Trp Arg Ser Glu Lys Glu Lys Ala Gln Asp Ala

325 330 335

Leu Leu Val Thr Val Arg Ile Gly Thr Asp Trp Val Val Ile Asp Val

340 345 350

Arg Gly Leu Leu Arg Asn Ala Arg Trp Arg Thr Ile Ala Pro Lys Asp

355 360 365

Ile Ser Leu Asn Ala Leu Leu Asp Leu Phe Thr Gly Asp Pro Val Ile

370 375 380

Asp Val Arg Arg Asn Ile Val Thr Phe Thr Tyr Thr Leu Asp Ala Cys

385 390 395 400

Gly Thr Tyr Ala Arg Lys Trp Thr Leu Lys Gly Lys Gln Thr Lys Ala

405 410 415

Thr Leu Asp Lys Leu Thr Ala Thr Gln Thr Val Ala Leu Val Ala Ile

420 425 430

Asp Leu Gly Gln Thr Asn Pro Ile Ser Ala Gly Ile Ser Arg Val Thr

435 440 445

Gln Glu Asn Gly Ala Leu Gln Cys Glu Pro Leu Asp Arg Phe Thr Leu

450 455 460

Pro Asp Asp Leu Leu Lys Asp Ile Ser Ala Tyr Arg Ile Ala Trp Asp

465 470 475 480

Arg Asn Glu Glu Glu Leu Arg Ala Arg Ser Val Glu Ala Leu Pro Glu

485 490 495

Ala Gln Gln Ala Glu Val Arg Ala Leu Asp Gly Val Ser Lys Glu Thr

500 505 510

Ala Arg Thr Gln Leu Cys Ala Asp Phe Gly Leu Asp Pro Lys Arg Leu

515 520 525

Pro Trp Asp Lys Met Ser Ser Asn Thr Thr Phe Ile Ser Glu Ala Leu

530 535 540

Leu Ser Asn Ser Val Ser Arg Asp Gln Val Phe Phe Thr Pro Ala Pro

545 550 555 560

Lys Lys Gly Ala Lys Lys Lys Lys Ala Pro Val Glu Val Met Arg Lys Asp

565 570 575

Arg Thr Trp Ala Arg Ala Tyr Lys Pro Arg Leu Ser Val Glu Ala Gln

580 585 590

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

595 600 605

Tyr Leu Lys Leu Ser Arg Arg Lys Glu Glu Leu Cys Arg Arg Ser Ile

610 615 620

Asn Tyr Val Ile Glu Lys Thr Arg Arg Arg Thr Gln Cys Gln Ile Val

625 630 635 640

Ile Pro Val Ile Glu Asp Leu Asn Val Arg Phe Phe His Gly Ser Gly

645 650 655

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

660 665 670

Arg Trp Phe Ile Gln Gly Leu His Lys Ala Phe Ser Asp Leu Arg Thr

675 680 685

His Arg Ser Phe Tyr Val Phe Glu Val Arg Pro Glu Arg Thr Ser Ile

690 695 700

Thr Cys Pro Lys Cys Gly His Cys Glu Val Gly Asn Arg Asp Gly Glu

705 710 715 720

Ala Phe Gln Cys Leu Ser Cys Gly Lys Thr Cys Asn Ala Asp Leu Asp

725 730 735

Val Ala Thr His Asn Leu Thr Gln Val Ala Leu Thr Gly Lys Thr Met

740 745 750

Pro Lys Arg Glu Glu Pro Arg Asp Ala Gln Gly Thr Ala Pro Ala Arg

755 760 765

Lys Thr Lys Lys Ala Ser Lys Ser Lys Ala Pro Pro Ala Glu Arg Glu

770 775 780

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

785 790 795 800

Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

805 810

<210> 3

<211> 40

<212> DNA

<213> sgRNA3 sequence

<400> 3

taatgtcgga acgctcaacg attgcccctc acgaggggac 40

<210> 4

<211> 29

<212> DNA

<213> sgRNA1 sequence

<400> 4

cgctcaacga ttgcccctca cgaggggac 29

<210> 5

<211> 18

<212> DNA

<213> sgRNA target sequence of Arabidopsis BRI1 gene

<400> 5

ctgcgaattc aatctccg 18

<210> 6

<211> 18

<212> DNA

<213> sgRNA target sequence of tobacco PDS gene

<400> 6

gtagtagcga ctccatgg 18

<210> 7

<211> 39

<212> DNA

<213> Artificial sequence U6-26p-F

<400> 7

ttcagaggtc tcttagtcga cttgccttcc gcacaatac 39

<210> 8

<211> 39

<212> DNA

<213> Artificial sequence U6-26t-R

<400> 8

agcgtgggtc tcgcgcctat tggtttatct catcggaac 39

<210> 9

<211> 44

<212> DNA

<213> Artificial sequence F2BRI1-U6-26t-F

<400> 9

ctgcgaattc aatctccgtt ttttttgcaa aattttccag atcg 44

<210> 10

<211> 39

<212> DNA

<213> Artificial sequences

<400> 10

cggagattga attcgcaggt cccctcgtga ggggcaatc 39

<210> 11

<211> 22

<212> DNA

<213> Artificial sequence F2BRI1crispr-identify-F

<400> 11

ctggcttcaa gtgctctgct tc 22

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence F2BRI1crispr-identify-R

<400> 12

gtaattcgcc ggaaaactcg 20

<210> 13

<211> 44

<212> DNA

<213> Artificial sequence F2NtPDS-U6-26t-F

<400> 13

gtagtagcga ctccatggtt ttttttgcaa aattttccag atcg 44

<210> 14

<211> 39

<212> DNA

<213> Artificial sequence F2NtPDS-gRNA-R

<400> 14

ccatggagtc gctactacgt cccctcgtga ggggcaatc 39

<210> 15

<211> 23

<212> DNA

<213> Artificial sequence NtPDS-seq-F

<400> 15

atgccccaaa ttggacttgt ttc 23

<210> 16

<211> 22

<212> DNA

<213> Artificial sequence NtPDS1-23-R

<400> 16

ctggagcgtg gacaacataa at 22

Claims (10)

1. CasF2 albumen, it is characterized in that, the aminoacid sequence of described CasF2 albumen is as shown in SEQ ID No.2; Or compared with the sequence shown in SEQ ID No.2, there is one or more amino acid replacement, deletion or addition, and have the same or similar biological functions. 2 . The gene encoding CasF2 protein according to claim 1 , wherein the nucleotide sequence of the encoding gene is shown in SEQ ID No.1. 3 . 3. An expression vector, characterized in that the expression vector comprises the nucleotide sequence of claim 2. 4. Use of the CasF2 protein of claim 1, the gene encoding the CasF2 protein of claim 2, or the expression vector of claim 3 in gene editing. 5. A CRISPR/Cas gene editing system, wherein the gene editing system comprises the CasF2 protein of claim 1 or a plasmid expressing the CasF2 protein. 6. The CRISPR/Cas gene editing system according to claim 5, wherein the gene editing system further comprises sgRNA or a plasmid expressing the sgRNA. 7. the application of the CRISPR/Cas gene editing system according to claim 5 or claim 6 in plant gene editing; Preferably, the plant is a monocotyledonous plant or a dicotyledonous plant; more preferably, the plant is selected from any one of Arabidopsis thaliana, tobacco, soybean and corn. 8. The application of claim 7, wherein the application comprises the steps of: (a) determining the sgRNA of the gene to be edited according to the PAM recognition site of the CasF2 protein; (b) cloning the gene sequence of the CasF2 protein and the corresponding sgRNA into an expression vector; (c) transforming the expression vector into Agrobacterium, and introducing the expression vector into a target plant through an Agrobacterium-mediated genetic transformation method to obtain a targeted gene editing plant; Preferably, in the genetic transformation, _MgCl2 is used to activate gene editing activity. 9 . The application according to claim 7 , wherein the plant is Arabidopsis thaliana, and the nucleotide sequence corresponding to the sgRNA is shown in SEQ ID No.3. 10 . 10 . The application according to claim 7 , wherein the plant is tobacco, and the nucleotide sequence corresponding to the sgRNA is shown in SEQ ID No.4. 11 .

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