Five Cas13d endonucleases and mediated RNA editing system thereof

Abstract

The invention discloses five Cas13d endonucleases and an RNA (Ribonucleic Acid) editing system mediated by the five Cas13d endonucleases. The Cas13d endonucleases are respectively Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4 and Gs13d-5, the amino acid sequences of the Cas13d endonucleases are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, and each endonucleases corresponds to a specific DR sequence. The invention also provides a protein-coding polynucleotide sequence optimized by an eukaryotic codon, which is used for efficiently expressing the Cas13d endonuclease, and in addition, the invention relates to high-efficiency crRNA (Complementary Ribonucleic Acid) targeting JEV (Japanese encephalitis virus). The Cas13d endonucleases and the crRNA can be used for preparing antiviral drugs, and the antiviral effect is achieved by regulating and controlling virus RNA. Experimental results show that the Cas13d endonuclease provided by the invention has high efficiency and specificity in the aspect of regulating and controlling RNA expression, and a new strategy and tool are provided for accurate editing of RNA.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to five kinds of Cas13d endonuclease and RNA editing systems mediated by the same. BACKGROUND

[0002] CRISPR (Clustered regularly interspaced short palindromic repeats) is a natural adaptive immune mechanism in bacteria and archaea. The CRISPR / Cas system is composed of two parts, one part is the CRISPR sequence, and the other part is the Cas protein (CRISPR associated protein) located near the CRISPR sequence. The CRISPR sequence is processed by the Cas protein to form a guide RNA (gRNA), and the gRNA combines with the Cas protein to form a complete CRISPR / Cas system. The commonly used Cas proteins at present are mainly Cas9 family, Cas12 family and Cas13 family. Among them, Cas9 family and Cas12 family are RNA-guided DNA nucleases, and Cas13 family is RNA-guided RNA nuclease.

[0003] The Cas13d protein has two signature HEPN domains and each Cas13d protein contains a conserved arginine and histidine residue dipeptide (R-XXXX-H) motif structure, and the two R-XXXX-H motif structures are the core of the RNA nuclease function of the Cas13d protein (Abudayyeh et al 2016). Different CRISPR / Cas13d systems have similar principles of action. When the bacteriophage infects the host bacteria, the host bacteria express the Cas13d protein and transcribe the CRISPR sequence into a long precursor CRISPR RNA (pre-crRNA), and the Cas13d protein combines with the pre-crRNA and processes it into mature crRNA to form a Cas13d-crRNA complex. Once the Cas13d-crRNA complex binds to the complementary single-stranded RNA (activator ssRNA) derived from the bacteriophage, a hybrid structure is formed between the crRNA spacer and the ssRNA, which causes the gradual change of the Cas13d protein conformation, and finally leads to the activation of the HEPN nuclease site (located on the outer surface of the protein). Mg 2+As a cofactor, the Cas13d protein is subsequently involved in RNA interference to cleave the activator ssRNA in cis and other available ssRNAs (phage and host ssRNA) in trans, and the trans-cleavage activity is also known as collateral cleavage activity, which is a feature in bacterial antiviral immunity by inducing host cell dormancy or programmed death to limit virus transmission.

[0004] Cas13d has multiple advantages such as relatively small amino acid sequence length, which makes it more easily packaged by adeno-associated virus (AAV) for in vivo delivery; no preference for pre-spacer sequence flanking site (PFS), which can target any RNA sequence; relatively high RNA cleavage activity, which ensures efficient interference of target gene expression. At present, there are still a large number of undiscovered CRISPR / Cas systems in the metagenomic database, and the RNA cleavage activity of these systems needs to be verified. Therefore, it is urgent to develop Cas13d endonuclease with high RNA editing activity. SUMMARY

[0005] The application develops five new Cas13d endonucleases Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4 and Gs13d-5, thereby establishing an efficient RNA editing system, and using CRISPR / Gs13d-2 technology to achieve inhibition of RNA virus infection.

[0006] In order to achieve the above purpose, the application adopts the following technical solutions:

[0007] The endonuclease in the CRISPR / Cas13d system comprises the following proteins:

[0008] I. Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4 and Gs13d-5 proteins of the amino acid sequences shown in SEQ ID NO. 1, 2, 3, 4 and 5;

[0009] II. Proteins having more than 80% sequence identity compared with the amino acid sequences shown in SEQ ID NO. 1, 2, 3, 4 and 5, and substantially retaining the biological function derived from the sequence;

[0010] III. Proteins having substitution, deletion or addition of one or more amino acids compared with the amino acid sequences shown in SEQ ID NO. 1, 2, 3, 4 and 5, and substantially retaining the biological function derived from the sequence.

[0011] The fusion protein comprises the above-mentioned Cas13d endonuclease, and a polypeptide connected to the N-terminus or C-terminus of the protein.

[0012] An isolated polynucleotide, which is a polynucleotide sequence encoding the above-mentioned Cas13d endonuclease, or a polynucleotide of the fusion protein.

[0013] Application of the above-mentioned Cas13d endonuclease in gene editing, including editing prokaryotes, eukaryotes or in vitro RNA.

[0014] A CRISPR / Cas13d gene editing system, comprising the above-mentioned Cas13d endonuclease, and a crRNA combined with the endonuclease, the crRNA sequence consisting of a Direct Repeat sequence and a Spacer Region sequence. Further, the Direct Repeat sequence of the Gs13d-1 protein is shown as SEQ ID NO. 11, the Direct Repeat sequence of the Gs13d-2 protein is shown as SEQ ID NO. 12, the Direct Repeat sequence of the Gs13d-3 protein is shown as SEQ ID NO. 13, the Direct Repeat sequence of the Gs13d-4 protein is shown as SEQ ID NO. 14, and the Direct Repeat sequence of the Gs13d-5 protein is shown as SEQ ID NO. 15.

[0015] A drug against Japanese encephalitis virus, comprising the above-mentioned Cas13d endonuclease and a crRNA targeting the target gene NS5. Preferably, the endonuclease is a Gs13d-3 protein with an amino acid sequence shown as SEQ ID NO. 3, and the spacer sequence in the crRNA sequence is shown as SEQ ID NO. 16.

[0016] The technical solution of the present application has the following main beneficial effects:

[0017] 1. The present application first provides a new member Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4 and Gs13d-5 of the CRISPR / Cas13d system family mined by combining metagenomics and experimental means.

[0018] 2. The present application first provides a Gs13d-2-mediated anti-RNA virus infection technical system. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1. Comparison of the amino acid sequence similarity of the guide RNA-dependent endonuclease Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 with known hfCas13d and UrCas13d and phylogenetic tree analysis. A. Comparison of the amino acid sequence similarity of Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 with known hfCas13d and UrCas13d; B. Phylogenetic tree analysis of Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 with known hfCas13d and UrCas13d.

[0020] Figure 2 . Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 and known hfCas13d gene locus and domain pattern diagram. A. Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 and known hfCas13d gene locus diagram; B. Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 and known hfCas13d domain diagram.

[0021] Figure 3 . Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 and known hfCas13d guide RNA DR sequence secondary structure folding (A) and multiple sequence alignment diagram (B).

[0022] Figure 4 . Targeted knockdown of 8 HEK293T endogenous genes to evaluate Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 cleavage RNA activity.

[0023] Figure 5 . Targeted knockdown of 4 HEK293T endogenous genes to evaluate Gs13d-1, Gs13d-2, Gs13d-4 cleavage RNA activity after changing the DR.

[0024] Figure 6 . Construction of hfCas13d plasmid vector connected with crRNA targeting JEV. A. Targeting JEV genome crRNA pattern diagram; B. Agarose gel electrophoresis identification of Bpil enzyme digestion CRISPR / hfCas13d plasmid vector; C. Agarose gel electrophoresis identification of successful connection of hfCas13d plasmid targeting JEV crRNA.

[0025] Figure 7. Screening of high-activity crRNA targeting JEV using CRISPR / hfCas13d targeting JEV-NS5.

[0026] Figure 8 . Evaluating and comparing the anti-JEV infection efficiency of Gs13d-2 and hfCas13d targeting JEV high-activity crRNA. DETAILED DESCRIPTION

[0027] TERMS

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0029] Genie scissor endonuclease family, where Genie means genie, representing bacterial origin, and scissor means gene scissors, indicating the gene editing function it may play. The corresponding Chinese name of Genie scissor endonuclease is "Lingjia" endonuclease, and Genie scissor gene editing system represents "Lingjia" endonuclease-mediated gene editing system, which is simply referred to as "Lingjia gene editing".

[0030] The application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the following examples without specific conditions are generally carried out according to the conventional conditions or according to the conditions recommended by the manufacturers.

[0031] Example 1. Mining of new guide RNA-dependent endonuclease based on metagenomics method

[0032] Based on the bioinformatics identification process of the new guide RNA-dependent endonuclease built by the inventor, the massive metagenomic sequencing data in the public databases such as NCBI nr (Non-Redundant Protein Sequence Database) non-redundant protein library, Global Microbial Gene Catalogue Database (GMGC) and the like are deeply mined for bacterial coding proteins. The approximate analysis process is as follows: for all contig sequences in the target database, the minced software is used to search and locate the CRISPR array, then the prodigal software is used to predict the proteins expressed adjacent to the CRISPR array, the CD-hit software is used to remove the redundancy of all the predicted proteins, the mega software is used for protein clustering analysis, the hmmer software is used for CRISPR-Cas similarity protein identification and classification, and finally five new unknown bacterial proteins are obtained, the amino acid sequences of which are shown as SEQ ID NO. 1, 2, 3, 4 and 5, and the nucleotide sequences of which are shown as SEQ ID NO. 6, 7, 8, 9 and 10.

[0033] Through phylogenetic evolutionary tree analysis, it is found that the five new bacterial proteins are located on different CRISPR / Cas13d system evolutionary branches Figure 1 B), and it is speculated that they may be new RNA-guided endonucleases. The present application names this kind of newly discovered protein from different bacteria as Genie scissor (GS) endonuclease. In order to facilitate subsequent research, the inventor further names the new unknown bacterial protein as Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4 and Gs13d-5 based on the bacterial species source, and the naming rule is "endonuclease + digital number".

[0034] Then, the inventor uses the localization blast program to perform sequence similarity comparison between the five newly discovered bacterial proteins and the NCBI nr database. As a result, it is found that the amino acid sequence similarity between the new Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4 and Gs13d-5 proteins and the known endonuclease hfCas13d is 43.40%, 45.74%, 48.35%, 51.61% and 45.87% Figure 1 A).

[0035] Further, the inventors analyzed the loci of the five proteins using CRISPR CasFinder software. The results showed that the five novel Cas13d endonucleases all have CRISPR array sequences, including multiple DR (Direct repeat) and spacer sequences, which are similar to the known hfCas13d. In addition, Gs13d-1, Gs13d-5 also have Cas1 protein, Gs13d-4 not only has Cas1 protein but also has Cas2 protein, Gs13d-1, Gs13d-2, Gs13d-3 also have WYL domain, which can enhance the cleavage activity of Cas13d Figure 2 A). Then the inventors named the DR sequences of hfCas13d, Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 as DR1, DR2, DR3, DR4, DR5, DR6, respectively, and predicted the secondary structure of DR1, DR2, DR3, DR4, DR5, DR6 through RNAfold web server (http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi) online website and multiple sequence alignment, the results showed that the DR of the five novel Cas13d endonucleases can form stem-loop structure Figure 3 A). Multiple sequence alignment of the DR sequences of the five novel Cas13d endonucleases with the known DR1 sequence showed that the DR sequences of the five novel Cas13d endonucleases were quite different from the known DR1 sequence, but the 3' end of the DR of the six Cas13d endonucleases was relatively conserved Figure 3 B), which may be related to the binding of crRNA to the 3' end.

[0036] In summary, it is speculated that the five novel Cas13d endonucleases may have RNA cleavage activity, which needs to be further verified by experiments.

[0037] Example 2. Guide RNA-dependent Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4, Gs13d-5 endonuclease has RNA cleavage activity

[0038] The present embodiment knocks down eight endogenous genes RPL4, CKB, RPS5, PPIA, EZH2, CA2, PPARG and B4GALNT1 in HEK293T cells through cell and molecular biology experiments to test the RNA cleavage activity of five novel Cas13d endonucleases. The crRNA paired with the target nucleic acid guides the Genie scissor protein to recognize and bind to the target nucleic acid, thereby triggering the cleavage activity of the Genie scissor protein to the target nucleic acid. The relative expression of the eight endogenous genes is detected by quantitative PCR to identify the RNA cleavage activity of the five novel Cas13d endonucleases.

[0039] The spacer sequence primers of the crRNA in the present embodiment are as follows (lowercase letters are linker sequences and uppercase letters are spacer sequences):

[0040]

[0041] The eight endogenous gene crRNA spacer sequence primers are cloned into linearized novel Cas13d and hfCas13d vectors by sticky end ligation. The sequence of the hfCas13d vector is available from addgene (#190034), and the novel Cas13d vector is obtained by replacing the hfCas13d sequence with the novel Cas13d sequence synthesized by Wuhan Tianyi Huayu Gene Technology Co., Ltd. HEK293T cells are inoculated, and the next day when the confluence is about 70%, the novel Cas13d and hfCas13d plasmids targeting the crRNA of the endogenous genes are delivered into the cells as the experimental group by transfection, and the empty plasmid of the corresponding nuclease is transfected as the control group. After 36 hours of transfection, the total RNA of the cells is extracted, and the expression levels of the eight endogenous genes in the experimental group compared with the control group are detected by quantitative PCR after reverse transcription. Taking the expression level of the endogenous genes in the experimental group less than 0.80 compared with the control group as the threshold, it is determined whether the novel Cas13d endonuclease has activity at this endogenous gene site. The results show that Gs13d-1 has activity at five endogenous genes, Gs13d-2 has activity at five endogenous genes, Gs13d-3 has activity at four endogenous genes, Gs13d-4 has activity at seven endogenous genes, and Gs13d-5 has activity at four endogenous genes. In addition, Gs13d-1 and Gs13d-2 have relatively high activity at the PPIA site, and Gs13d-3 has relatively high activity at the PPIA and RPL4 sites. Figure 4 In summary, the five novel Cas13d endonucleases all have RNA cleavage activity, and Gs13d-1, Gs13d-2 and Gs13d-4 with relatively more active sites are selected for further research.

[0042] Example 3. Identification of the effect of different DR sequences on the RNA cleavage activity of novel Cas13d endonuclease

[0043] This example identifies the effect of different DR sequences on the RNA cleavage activity of novel Cas13d endonuclease by cell and molecular biology experiments. First, the DR1 sequence on the Gs13d-1, Gs13d-2, Gs13d-4 plasmid was changed to the original DR sequence of the three nucleases (Gs13d-1-DR2, Gs13d-2-DR3, Gs13d-4-DR5). Gs13d-1-DR2, Gs13d-2-DR3, Gs13d-4-DR5 plasmids targeting EZH2, PPIA, RPL4, B4GALNT1 crRNA were delivered to HEK293T cells by transfection as the experimental group, and Gs13d-1-DR2, Gs13d-2-DR3, Gs13d-4-DR5 empty plasmids were transfected at the same time as the control group. After 36h of transfection, total RNA was extracted from the cells, and the expression levels of the four endogenous genes in the experimental group relative to the control group were detected by fluorescence quantitative PCR after reverse transcription. The results show that the knockdown efficiency of Gs13d-1-DR2 on EZH2, PPIA, B4GALNT1 is significantly higher than that of Gs13d-1-DR1, and there is no significant change in the knockdown efficiency of Gs13d-1 on RPL4 before and after changing DR Figure 5 A); the knockdown efficiency of Gs13d-2-DR3 on the four endogenous genes is significantly higher than that of Gs13d-2-DR1 Figure 5 B); the knockdown efficiency of Gs13d-4-DR5 on EZH2 is significantly higher than that of Gs13d-4-DR1, and the knockdown efficiency on RPL4 is significantly lower than that of Gs13d-4-DR1, and there is no significant change in the knockdown efficiency of Gs13d-4 on PPIA, B4GALNT1 before and after changing DR Figure 5 C). After changing the DR of the three novel Cas13d endonucleases, Gs13d-2-DR3 has the best knockdown effect on the four endogenous genes of HEK293T cells, so Gs13d-2-DR3 is selected for further study.

[0044] Example 4. Screening of high-activity crRNA targeting and inhibiting JEV infection using CRISPR / hfCas13d system

[0045] This example screens high-activity crRNA targeting and inhibiting Japanese encephalitis virus (JEV) infection using the CRISPR / hfCas13d system. Five JEV-targeting crRNAs were designed using the JEV genome data measured by the laboratory earlier, and the spacer sequence primers of the crRNAs are as follows (lowercase letters are linker sequences, and uppercase letters are spacer sequences):

[0046]

[0047] The above-mentioned crRNA spacer sequence primer pairs were cloned into the linearized hfCas13d vector using a sticky-end ligation method. Sanger sequencing results showed that all crRNAs were successfully ligated. After plasmid extraction, agarose gel electrophoresis was performed, and the plasmid bands were relatively uniform. The above results indicate that the CRISPR / hfCas13d plasmid vector targeting the JEV genome was successfully constructed. Figure 6 ).

[0048] HEK293T cells were then inoculated. The next day, when confluence reached approximately 70%, the hfCas13d vector, linked to JEV-targeting crRNA, was transfected into HEK293T cells. Simultaneously, the empty hfCas13d vector was transfected as a control. After 24 hours, the JEV-NS5 viral gene plasmid was transfected twice. 24 hours after the second transfection, total RNA was extracted from the cells, and the expression level of the viral gene plasmid was detected by reverse transcription and quantitative real-time PCR. The results showed that JEV-crRNA1 had the best inhibitory effect on the expression of the JEV-NS5 viral gene plasmid. Figure 7 ).

[0049] Therefore, JEV-crRNA1 was selected for the challenge experiment. Huh7 cells were inoculated, and on the second day, when confluence reached approximately 70%, the CRISPR / hfCas13d plasmid vector linked to JEV-crRNA1 was transfected into the JEV-susceptible Huh7 cell line as the experimental group. Simultaneously, the empty CRISPR / hfCas13d vector was transfected as a control. JEV infection occurred 36 hours after transfection. Total RNA was extracted from the cells 24 hours later, and the difference in JEV expression levels between the experimental and control groups was detected by reverse transcription and quantitative real-time PCR. The results showed that hfCas13d combined with JEV-crRNA1 significantly resisted JEV infection.

[0050] Example 5. Targeted inhibition of JEV virus infection using a novel CRISPR / Gs13d-2 system.

[0051] The present embodiment utilizes the CRISPR / Gs13d-2 system to inhibit JEV infection. First, the inhibitory effect of Gs13d-2 nuclease on JEV proliferation is evaluated, and the high-activity JEV-crRNA1 clone screened in the previous stage is cloned into the linearized Gs13d-2-DR3 vector, and the Gs13d-2-DR3 plasmid connected with JEV-NS5 crRNA1 is transfected into Huh7 cells as the experimental group, and the Gs13d-2-DR3 empty plasmid is transfected as the control group. After 36 hours of transfection, the Huh7 cells are infected with JEV, and the total RNA of the cells is extracted 24 hours after infection. After reverse transcription, the JEV proliferation level is detected by fluorescence quantitative PCR, and the results show that the JEV proliferation in the experimental group is significantly inhibited compared with the control group Figure 8

[0052] To compare the antiviral efficiency difference between Gs13d-2 and hfCas13d, the Gs13d-2-DR3 plasmid is transfected as described above, and the hfCas13d plasmid connected with JEV-crRNA1 and the hfCas13d empty plasmid are transfected at the same time, and the above Gs13d-2-DR3 plasmid is also infected with JEV. The total RNA of the cells is extracted 24 hours after infection, and the JEV proliferation level is detected by fluorescence quantitative PCR after reverse transcription. The results show that the inhibitory effect of the developed Gs13d-2 on JEV proliferation is better than that of hfCas13d Figure 8 Based on the above experiments, it is shown that the RNA editing system based on the novel Cas13d endonuclease is successfully established.​

Claims

1. An endonuclease in a CRISPR / Cas13d system, characterized in that, The proteins include: I. Gs13d-1, Gs13d-2, Gs13d-3, Gs13d-4 and Gs13d-5 proteins with amino acid sequences shown in SEQ ID NO. 1, 2, 3, 4 and 5; II. Proteins with more than 80% sequence identity compared with the amino acid sequences shown in SEQ ID NO. 1, 2, 3, 4 and 5, and substantially retaining the biological function derived from the sequences; III. Proteins with substitution, deletion or addition of one or more amino acids compared with the amino acid sequences shown in SEQ ID NO. 1, 2, 3, 4 and 5, and substantially retaining the biological function derived from the sequences.

2. A fusion protein, characterized in that, The proteins include:

3. A polynucleotide, characterized in that, The polynucleotide is a polynucleotide encoding the endonuclease of claim 1, or a polynucleotide encoding the fusion protein of claim 2.

4. The polynucleotide of claim 3, wherein, The polynucleotide sequence encoding the Gs13d-1 protein is shown in SEQ ID NO. 6, the polynucleotide sequence encoding the Gs13d-2 protein is shown in SEQ ID NO. 7, the polynucleotide sequence encoding the Gs13d-3 protein is shown in SEQ ID NO. 8, the polynucleotide sequence encoding the Gs13d-4 protein is shown in SEQ ID NO. 9, and the polynucleotide sequence encoding the Gs13d-5 protein is shown in SEQ ID NO.

10.

5. Vector, characterized in that, The vector comprises the polynucleotide of claim 3 or 4.

6. A host cell characterized in that, The host comprises the polynucleotide of claim 3 or 4, or the vector of claim 5.

7. Use of the endonuclease of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3 or 4, or the vector of claim 5, or the host cell of claim 6 in gene editing.

8. Use according to claim 7, characterized in that, The gene editing includes editing prokaryotes, eukaryotes or in vitro RNA.

9. A CRISPR / Casl3d gene editing system, characterized in that, The endonuclease of claim 1, or the fusion protein of claim 2, or the polynucleotide of claim 3 or 4, or the vector of claim 5, or the host cell of claim 6.

10. The CRISPR / Casl3d gene editing system of claim 9, wherein, The crRNA capable of binding to the endonuclease of claim 1, the crRNA sequence consisting of a direct repeat sequence and a spacer sequence.

11. The CRISPR / Casl3d gene editing system of claim 10, wherein, The direct repeat sequence of the Gs13d-1 protein is shown in SEQ ID NO. 11, the direct repeat sequence of the Gs13d-2 protein is shown in SEQ ID NO. 12, the direct repeat sequence of the Gs13d-3 protein is shown in SEQ ID NO. 13, the direct repeat sequence of the Gs13d-4 protein is shown in SEQ ID NO. 14, and the direct repeat sequence of the Gs13d-5 protein is shown in SEQ ID NO.

15.

12. A medicament against Japanese encephalitis virus, characterized by comprising the compound of claim 1. The endonuclease of claim 1 and the crRNA targeting the target gene NS5.

13. The medicament for use against Japanese encephalitis virus according to claim 12, wherein The endonuclease is a Gs13d-3 protein with an amino acid sequence shown in SEQ ID NO. 3, and the spacer sequence in the crRNA sequence is shown in SEQ ID NO.

16. The endonuclease is a Gs13d-3 protein with an amino acid sequence shown in SEQ ID NO. 3, and the spacer sequence in the crRNA sequence is shown in SEQ ID NO. 16.

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