CrRNA for knocking down mRNA of zebrafish, gene editing system and preparation and application of crRNA

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to crRNA for knocking down zebrafish mRNA, a gene editing system and preparation and application of the crRNA. The nucleotide sequence of the crRNA is as shown in SEQ ID NO. 11. The gene editing system is a CRISPR-hfCas13x (clustered regularly interspaced short palindromic repeats-hfCas13x) editing system and comprises crRNA (ribonucleic acid) and zCas13x The invention also provides a method for knocking down the mRNA of the zebrafish based on the CRISPR-hfCas13x editing system. The method comprises the following steps: transcribing a zCas13x target gene plasmid into mRNA; the method comprises the following steps: designing and synthesizing a crRNA sequence of zebra fish endogenous gene mRNA; the zCas13x mRNA and the crRNA are microscopically injected into fertilized eggs of the zebrafish, so that the mRNA of the zebrafish is knocked down. The method has the characteristics of high specificity and high safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a crRNA for knocking down zebrafish mRNA, a gene editing system, and preparation and application thereof. Background Art

[0002] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) is a defense mechanism against external genetic material formed by bacteria and archaea during their long-term evolution. It mainly uses CRISPR RNAs (crRNA) to guide nucleases to resist the invasion of external nucleic acids. The CRISPR system integrates fragments of invading phage and plasmid DNA into its own CRISPR, and uses the corresponding CRISPR RNAs (crRNA) to guide the breakage and degradation of homologous sequences, thereby providing immune defense.

[0003] CRISPR-Cas13 is a member of the CRISPR-Cas system family. Unlike CRISPR-Cas9, which mainly targets DNA, CRISPR-Cas13 mainly targets RNA. The CRISPR-Cas13 system specifically binds to the target RNA sequence through a specific crRNA, and then the Cas13 protein recognizes and cuts these complementary target RNA sequences and activates non-specific RNA degrading enzymes, leading to the degradation of the target RNA. Existing studies have shown that CRISPR-Cas13 technology has the following characteristics: 1) Targeting RNA: CRISPR-Cas13 is mainly used to target RNA, which gives it advantages in editing RNA viruses, regulating gene expression, and studying RNA functions. 2) High specificity: Through the rational design of sgRNA, CRISPR-Cas13 can achieve highly specific editing of target RNA and reduce the impact on non-target RNA. 3) Responsiveness: The Cas13 protein generates a "signal" when cutting the target RNA, which makes CRISPR-Cas13 technology potential in the field of detection and diagnosis. 4) No DNA transfection required: Compared with CRISPR-Cas9, CRISPR-Cas13 does not require the introduction of gene editing tools into the cell nucleus, so it does not involve the complex process of the cell nucleus. The CRISPR-Cas13 system provides new possibilities for the study and treatment of RNA-related diseases.

[0004] CRISPR-Cas13x, also known as Cas13e, is a new type of CRISPR-Cas13 system discovered through computational analysis of large-scale microbial metagenomic data. As a new type of RNA editing tool, it can be widely used in RNA knockdown, RNA single-base editing, RNA site-directed modification, RNA live cell tracing, and nucleic acid detection. Compared with traditional RNA interference technology, the CRISPR-Cas13x system has more advantages. First, a significant advantage of CRISPR-Cas13x is its small size. Cas13X.1 (Cas13e.1) has a volume of only 775 amino acids and is one of the smallest Cas13 proteins known to date. The ultra-small size enables CRISPR-Cas13x to be packaged in a single AAV (adeno-associated virus) vector for in vivo delivery, solving the problem that the previous Cas13 protein was large and difficult to deliver. Second, the CRISPR-Cas13x system has high specificity and safety, low off-target rate, and low side-cutting effect. First, it has no PFS (the protospacer flanking sequence) region in function, and can selectively target various regions of the target mRNA; second, it has no trans-cutting effect during the process of mRNA shearing and editing, and has high specificity for mRNA targeting. In summary, CRISPR-Cas13x has higher efficiency and specificity, better safety and stability than traditional RNA interference technology. These advantages make CRISPR-Cas13x an important mRNA editing tool and can be developed as a practical tool for biological mRNA editing.

[0005] CRISPR Cas13 is a type of editing system that can target RNA. Currently, the system is increasingly being used for gene knockdown in various species. Researchers have used Cas13d and Cas13a in zebrafish to verify the knockdown effect, and there are also literature reports that Cas13x can play a role in RNA in mammalian cells and mice. For example, patent CN114231561A discloses a method for knocking down animal mRNA based on CRISPR-Cas13d and its application. It transcribes the Cas13d target gene plasmid into mRNA; designs and synthesizes the crRNA sequence of the animal's endogenous gene mRNA; and micro-injects Cas13d mRNA and crRNA into the animal's fertilized eggs, so that the CRISPR-Cas13d editing system can mediate the directional shearing of the target mRNA in the animal through crRNA and knock it down. This invention uses zebrafish as experimental animals and achieves the technical goal of directly knocking down the target mRNA in the animal by using the CRISPR-Cas13d editing system. However, no studies have been found using CRISPRhfCas13x for gene knockdown in zebrafish.

[0006] hfcas13x (high-fidelity Cas13x) is an engineered CRISPR-Cas13 system and an improved version of Cas13x. Through engineering, it improves the specificity and safety of RNA editing and reduces off-target effects. Therefore, it has greater application potential in the field of RNA editing and gene therapy, providing a new tool for future gene therapy. Summary of the invention

[0007] In view of this, the present invention proposes a crRNA targeting endogenous genes in zebrafish, and a CRISPR-hfCas13x editing system for knocking down zebrafish mRNA. At the same time, a method for knocking down zebrafish mRNA based on CRISPR-hfCas13x is established. This method is efficient and convenient, and is of great significance for the study of gene function in zebrafish and humans, and has practical significance in the treatment of certain diseases and the development of drugs for the treatment of related diseases.

[0008] One of the purposes of the present invention is to provide a crRNA targeting endogenous genes in zebrafish.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A crRNA targeting an endogenous gene of zebrafish, wherein the nucleotide sequence of the crRNA is shown in SEQ ID NO.11.

[0011] Further, the endogenous gene is the Ntl gene.

[0012] Furthermore, the crRNA targets the front segment of the first exon of the zebrafish Ntl gene; the nucleotide sequence of the front segment of the first exon is shown in SEQ ID NO.2; the zebrafish targeting sequence spacer is shown in SEQ ID NO.10.

[0013] A second object of the present invention is to provide a CRISPR-hfCas13x editing system containing the aforementioned crRNA.

[0014] To achieve the above object, the present invention adopts the following technical solutions:

[0015] CRISPR-hfCas13x editing system containing the aforementioned crRNA.

[0016] Further, the system includes the aforementioned crRNA and zCas13x mRNA; the sequence of the zCas13x mRNA is shown in SEQ ID NO.17.

[0017] A third object of the present invention is to provide a method for constructing the aforementioned CRISPR-hfCas13x editing system.

[0018] To achieve the above object, the present invention adopts the following technical solutions:

[0019] The method for constructing the aforementioned CRISPR-hfCas13x editing system comprises the following steps:

[0020] (1) Optimizing the target gene sequence encoding hfCas13x by zebrafish codon preference and synthesizing a zCas13x plasmid, wherein the zCas13x plasmid is transcribed in vitro to obtain zCas13x mRNA;

[0021] (2) Design and synthesize crRNA of endogenous zebrafish gene mRNA.

[0022] Furthermore, the method is not only applicable to the construction of a CRISPR-hfCas13x editing system for knocking down zebrafish mRNA, but can also be optimized in other species, including but not limited to fish, mice, rabbits, cattle, and sheep.

[0023] Further, in step (1), the sequence of hfCas13x after zebrafish codon optimization is shown in SEQ ID NO.1.

[0024] Further, in step (1), the synthesis of the zCas13x plasmid is to clone the optimized gene coding sequence into the pcs2+ plasmid vector to obtain the zCas13x plasmid, and the plasmid vector is exogenously inserted with the zCas13x gene sequence.

[0025] Preferably, the plasmid vector also includes an SP6 promoter, an ori replicon and an ampicillin resistance gene sequence.

[0026] More preferably, the element sequence of the zCas13x plasmid is: SP6 Promter-zCas13x-SV40 ploy(A)signal-Amp-Amp Promoter.

[0027] As a preferred solution, in step (1), the synthesis and transcription of the zCas13x plasmid are specifically as follows:

[0028] Step A: Obtain linearized zCas13x plasmid using restriction endonuclease digestion;

[0029] Step B: The linearized zCas13x plasmid obtained in step A was transcribed in vitro using the SP6 RNA transcription kit, and zCas13x mRNA was purified.

[0030] Preferably, in step A, the plasmid enzyme digestion system includes: 1 μL restriction endonuclease, 1 μL (1 μg) zCas13x plasmid, 5 μL restriction endonuclease buffer, 43 μL ddH2O, a total of 50 μL.

[0031] Preferably, the reaction conditions of step A are: enzyme digestion in a 37°C water bath for 30 min.

[0032] Preferably, in step B, the transcription reaction system includes: 6 μL (concentration of 100 ng / μL) linearized zCas13x plasmid, 2 μL SP6 RNA enzyme mixture, 2 μL transcription buffer, 10 μL NTP mixture, and the transcription reaction system is 20 μL in total.

[0033] Preferably, in step B, the transcription reaction conditions are: incubation at 37° C. for 2.5 h to 3 h.

[0034] Further, step (2) includes the following steps:

[0035] 1) synthesizing primers for constructing crRNA transcription template DNA; the primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO.12 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.13;

[0036] 2) using the primers obtained in step 1) to perform PCR amplification to obtain a template DNA of crRNA;

[0037] 3) Using the DNA sequence obtained in step 2) as a transcription template, in vitro transcription is performed and crRNA is purified.

[0038] Wherein, the forward primer carries a T7 promoter, and the reverse primer carries a target site.

[0039] Preferably, in step 2), the PCR reaction system includes: 5 μL forward primer, 5 μL reverse primer, 25 μL ordinary taqDNA polymerase Mix (2x GO taq master Mix), 15 μL ddH2O, a total of 50 μL.

[0040] Preferably, in step 2), the PCR reaction conditions are: pre-denaturation at 94°C for 4 min, followed by a 3-step cycle reaction: 94°C-30 s, 60°C-40 s, 72°C-30 s for a total of 30 cycles, then 72°C-10 min, and finally keeping warm at 4°C.

[0041] Preferably, in step 3), the transcription reaction system includes: 6 μL (concentration is 100 ng / μL) template DNA, 2 μL ATP, 2 μL GTP, 2 μL UTP, 2 μL CTP, 2 μL T7 transcriptase, 2 μL transcription buffer, 2 μL ddH2O, a total of 20 μL.

[0042] Preferably, in step 3), the transcription reaction system is mixed and placed in a PCR instrument for incubation at 37°C for 4 hours. After the incubation is completed, samples are taken to observe whether there are RNA bands. After verification, DNase I enzyme is added to the system to remove DNA fragments in the reaction system, and the incubation is continued for 15 minutes. After the transcription is completed, the obtained crRNA is purified using the Adelaide RNA purification kit.

[0043] A fourth object of the present invention is to provide a method for knocking down zebrafish mRNA using the aforementioned CRISPR-hfCas13x editing system.

[0044] To achieve the above object, the present invention adopts the following technical solutions:

[0045] The method of knocking down zebrafish mRNA using the aforementioned CRISPR-hfCas13x editing system is to microinject the zCas13xmRNA and the crRNA into zebrafish fertilized eggs and cultivate them, so that the CRISPR-hfCas13x editing system can direct the shearing of the target mRNA in the zebrafish through crRNA-mediated directional shearing and knockdown.

[0046] Furthermore, the fertilized egg is a 1-cell stage fertilized egg.

[0047] Furthermore, the usage ratio of zCas13x mRNA and crRNA is 2 to 3:1, preferably 2.5:1.

[0048] Furthermore, the zCas13x mRNA, the crRNA and phenol red were mixed and co-injected into zebrafish fertilized eggs.

[0049] Preferably, the final concentration of phenol red is 5%.

[0050] Furthermore, the cultivation temperature is 26-28°C.

[0051] Furthermore, real-time fluorescence quantitative PCR detection was used to determine the mRNA targeting efficiency, knockdown status and embryonic phenotype.

[0052] Preferably, the front primer for detecting Ntl gene expression by real-time fluorescence quantitative PCR is shown as SEQ ID NO.15, and the rear primer is shown as SEQ ID NO.16.

[0053] A fifth object of the present invention is to provide an application of the aforementioned crRNA and / or the aforementioned CRISPR-hfCas13x editing system in the preparation of a kit for knocking down zebra mRNA.

[0054] The beneficial effects of the present invention are:

[0055] 1. The present invention provides a technical method for knocking down zebrafish target mRNA by using the technology of micro-coinjection of zCas13x mRNA and crRNA, and directly shearing and knocking down the target mRNA in zebrafish by crRNA-mediated directional direct use of the CRISPR-hfCas13x editing system. The present invention applies the CRISPR-hfCas13x technology to the in vivo experimental process of zebrafish for the first time, and successfully transforms the CRISPR-hfCas13x system into a practical tool that works in zebrafish.

[0056] 2. The CRISPR-hfCas13x editing system provided by the present invention and the method for knocking down zebrafish mRNA based on the system are significantly different from the traditional method for knocking down mRNA. The traditional method for knocking down mRNA uses different CRISPR-Cas systems, and its shearing effect has target selectivity and shearing non-specificity; while the present invention eliminates the limitation of gene mRNA targeting sites, does not produce non-specific targeting effects during the targeting process, and can randomly select mRNA targeting sites to obtain higher mRNA targeting efficiency.

[0057] 3. The targeting of the present invention in zebrafish is highly specific, eliminating the shearing and interference of ribonucleases in the CRISPR system on non-target sites; the entire operation implementation process is simple and effective.

[0058] 4. The present invention achieves the technical goal of directly knocking down target mRNA in zebrafish using the CRISPR-hfCas13x editing system, which is helpful for the treatment of early gene expression diseases in humans and other organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The figure shows the expression of Ntl gene mRNA in the microinjection 6hpf experimental group and the control group;

[0060] Figure 2 This is a diagram of the phenotype of the microinjected 24hpf control group zebrafish;

[0061] Figure 3 This is a diagram of the phenotype of zebrafish in the microinjection experimental group at 24hpf. DETAILED DESCRIPTION

[0062] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0063] The present invention proposes a technical method for practical application of crRNA-mediated CRISPR-hfCas13x editing technology in animals to knock out zebrafish mRNA, but it is not limited to zebrafish species, and other species can also be selected for optimization, such as fish, mice, rabbits, cattle, sheep, etc.

[0064] Example 1. Synthesis and transcription of zCas13x gene plasmid vector

[0065] Search the target gene sequence of CRISPRVI-X type (hfCas13x) on the addgene website, and optimize the zebrafish codon preference of the obtained hfCas13 original sequence without changing the encoded amino acid. The gene sequence of hfCas13 zebrafish codon optimization is shown in SEQ ID NO.1. The target gene zCas13x was chemically synthesized by Shanghai Sangon Biotechnology Co., Ltd. After obtaining the optimized gene coding sequence, its gene sequence was cloned into the pcs2+ plasmid vector, and the element order was: SP6 Promter-zCas13x-SV40 ploy(A)signal-Amp-Amp Promoter. The specific steps are as follows:

[0066] 1. The linearized zCas13x plasmid was obtained by digestion with restriction endonuclease (NotⅠ) (Takara QuickCut Enzyme). The entire plasmid digestion system was 50 μL. Various reagents were added in the order shown in Table 1, mixed with an oscillator, centrifuged instantaneously, and placed in a 37°C water bath for digestion for 30 minutes. The digestion products of the vector were subjected to agarose gel electrophoresis to recover and purify the brighter target bands.

[0067] Table 1. Plasmid restriction enzyme digestion system

[0068] Reagents Volume (μL) <![CDATA[ddH2O]]> 43 10× Endonuclease Buffer 5 Plasmids 1(1μg) NotⅠ restriction enzyme 1 total 50

[0069] 2. In vitro transcription of zCas13x gene sequence

[0070] SP6 RNA transcription kit (mMESSAGE The linearized zCas13x plasmid obtained in the first step was transcribed in vitro using Kit, Life Technologies. The entire reaction system was 20 μL. The relevant reagents were gradually added to the RNase-free PCR tube in the order shown in Table 2. After mixing, the mixture was gently shaken with an oscillator. After instant centrifugation, the tube was placed in a PCR instrument for transcription experiments.

[0071] Table 2. In vitro transcription reaction system

[0072] Reagents Volume (μL) 10×TranscriptionBuffer 2 zCas13x linear plasmid template 6(600ng) SP6 RNase Mix 2 2×NTPMix 10 total 20

[0073] Place the mixed 20 μL transcription system in a PCR instrument and incubate at 37°C for 2.5 h to 3 h, then take 1 μL for agarose gel electrophoresis verification to observe whether there are RNA bands; after verification, add 1 μL of DNase to the system and continue incubation for 15 min.

[0074] After transcription is completed, the obtained mRNA is purified using the lithium chloride reagent in the SP6 kit: 30 μL of enzyme-free water and an equal volume (30 μL) of LiCl solution are added to the reaction system, and after sufficient mixing, precipitation is carried out at -80°C for more than 30 minutes, and after precipitation, centrifugation is carried out at 4°C 15000rpm for 15 minutes, and the waste liquid is discarded. 1 mL of 75% anhydrous ethanol is added to the precipitate, and the precipitate is washed and centrifuged at 4°C 15000rpm for 15 minutes, and the waste liquid is discarded, and centrifugation is carried out at 4°C 15000rpm for 15 minutes again, and the waste liquid is discarded. After drying, 30 μL of enzyme-free water is added to resuspend the mRNA precipitate, and the mRNA concentration is measured and stored in an RNase-free centrifuge tube at -80°C. The nucleotide sequence of zCas13xmRNA obtained in this example is shown in SEQ ID NO.17.

[0075] Example 2. Design and synthesis of crRNA

[0076] 1. Design new crRNA sequences

[0077] According to the design principle of crRNA, the zebrafish Ntl gene was selected as the mRNA sequence of the target gene, and the designed target site was in the first exon front section of Ntl (exon 1). Then the primers of crRNA were designed and synthesized. The sequence information is shown in Table 3.

[0078] Table 3. Sequence information table

[0079]

[0080] 2. Template DNA for synthesizing crRNA

[0081] The designed Forward primer and Reverse primer were used for PCR, and the PCR reaction system is shown in Table 4:

[0082] Table 4. PCR reaction system

[0083] Reagents Volume (μL) <![CDATA[ddH2O]]> 15 Forwardprimer 5 Reverseprimer 5 2xGOtaqmasterMix 25 total 50

[0084] The PCR reaction conditions were as follows: pre-denaturation at 94°C for 4 min, 3-step cycle reaction (94°C for 30 s, 60°C for 40 s, 72°C for 30 s, 30 cycles in total), then 72°C for 10 min, and finally incubation at 4°C; after electrophoresis detection of the PCR product, purification was performed.

[0085] 3. Transcribe and synthesize the targeting crRNA of the Ntl gene

[0086] The DNA sequence synthesized in the previous step was used as a reaction template to transcribe into RNA, and in vitro transcription was performed using a T7 transcription kit (Vazyme T7 High Yield RNA Transcription kit). The transcription system is shown in Table 5:

[0087] Table 5. Transcription reaction system

[0088] Reagents Volume (μL) <![CDATA[ddH2O]]> 2 5×TranscriptionBuffer 2 Template DNA 6(600ng) ATP 2 GTP 2 CTP 2 UTP 2 T7 transcriptase 2 total 20

[0089] The reaction mixture was placed in a PCR instrument for transcription and incubated at 37°C for 4 hours. After the incubation was completed, 1 μL was taken for agarose gel electrophoresis verification. After the verification was correct, DNase I enzyme was added to the crRNA product to remove the DNA fragments in the reaction system, and incubated at 37°C in a PCR instrument for 15 minutes.

[0090] Purification of RNA (Adlai Kit RNAclean RNA Cleaning Purification Kit, Catalog No.: RN14): Add RNase-Free water to the RNA sample, make up to 100μL, add 350μL solution RC, and mix thoroughly. Add 250μL anhydrous ethanol, mix thoroughly, and proceed to the next step immediately. Transfer the solution and precipitate obtained in the previous step to the adsorption column RA, centrifuge at 12000rpm for 30 seconds, and discard the waste liquid in the collection tube. Add 500μL of rinsing solution RW with ethanol added to the adsorption column RA, leave it at room temperature for 2min, centrifuge it at 12000rpm for 30 seconds, discard the waste liquid, and place RA in the collection tube. Centrifuge at 12000rpm for 2min to remove the residual liquid. Transfer the adsorption column RA to a new centrifuge tube, add 30μL RNase-Free water, leave it at room temperature for 2min, centrifuge it at 12000rpm for 2min, obtain crRNA, and store it at -80℃.

[0091] Example 3. Microinjection of fertilized zebrafish eggs

[0092] 1. Obtaining fertilized zebrafish embryos

[0093] Sexually mature male and female parent zebrafish are placed in a mating and spawning box at a ratio of 2:1 and cultured with partitions; the mating and spawning box is placed in a constant temperature and quiet environment at 26-28°C in the dark overnight, and the light cycle is 14h day:10h dark; on the second day, after the dark cycle is over, the partition is opened, the male and female parent zebrafish are mixed, and after the parent fish lay eggs for 10-15 minutes, the parent fish are taken out, the fertilized eggs gathered at the bottom of the spawning box are sucked out and arranged sequentially on an agarose fixing plate for microinjection.

[0094] 2. Microinjection of zCas13x and Ntl

[0095] The transcribed zCas13x mRNA, Ntl crRNA and phenol red were mixed at final concentrations of 200 ng / μL, 80 ng / μL and 5%; then co-injected into 1-cell stage zebrafish fertilized egg embryos; after the injection, the obtained fertilized egg embryos were placed in an environment of 26-28°C for development.

[0096] 3. Real-time fluorescence quantitative PCR was used to detect the expression of Ntl gene mRNA.

[0097] The injected zebrafish fertilized eggs developed to 24 hpf, and embryos from the control group and the experimental group were taken, 3 groups each, 30 eggs in each group. The genomic RNA of the 3 groups of zebrafish eggs was extracted, and the expression changes of Ntl were detected by real-time fluorescence quantitative PCR (Realtime-PCR). The front primer of real-time fluorescence quantitative PCR detection is shown in SEQ ID NO.15, and the rear primer is shown in SEQ ID NO.16. The results are shown in Figure 1 As shown, it can be seen that the experimental group can reduce the expression of Ntl gene at 6hpf.

[0098] 4. Phenotypic statistics of Ntl mRNA knockdown

[0099] After the microinjected zebrafish embryos developed for one day, the deformities of the spine and tail of all embryos in each group were counted, and the knockdown phenotype rate of each group of zebrafish embryos was calculated. Figure 2 , Figure 3 As shown in Table 6, it can be seen that the deformity rate in the experimental group was significantly higher than that in the control group.

[0100] Table 6. Statistical results of developmental phenotypes in the microinjection 24 hpf experimental group and control group

[0101] combination Total sample size Normal sample size Abnormal sample size Deformity rate ZCas13XmRNA+NtlcRNA 87 14 73 83.91％ ZCas13XmRNA 117 97 20 17.09％ NtlcrRNA 96 81 15 15.63％ Control 201 198 3 1.49％

Claims

1. A crRNA targeting an endogenous gene of zebrafish, characterized in that: The nucleotide sequence of the crRNA is shown in SEQ ID NO.

11.

2. The crRNA according to claim 1, characterized in that The endogenous gene is the Ntl gene.

3. The crRNA according to claim 1, characterized in that The crRNA targets the first exon front segment of the zebrafish Ntl gene; the nucleotide sequence of the first exon front segment is shown in SEQ ID NO.2; the zebrafish targeting sequence spacer is shown in SEQ ID NO.

10.

4. A CRISPR-hfCas13x editing system comprising the crRNA described in any one of claims 1 to 3.

5. The system according to claim 4, characterized in that The system comprises the crRNA and zCas13x mRNA according to any one of claims 1 to 3; the sequence of the zCas13x mRNA is shown in SEQ ID NO.

17.

6. The method for constructing the CRISPR-hfCas13x editing system according to any one of claims 4 to 5, characterized in that The steps include: (1) Optimizing the target gene sequence encoding hfCas13x by zebrafish codon preference and synthesizing a zCas13x plasmid, wherein the zCas13x plasmid is transcribed in vitro to obtain zCas13x mRNA; (2) Design and synthesize crRNA of endogenous zebrafish gene mRNA.

7. The method according to claim 6, characterized in that In step (1), the sequence of hfCas13x after zebrafish codon optimization is shown in SEQ ID NO.

1.

8. The method according to claim 6, characterized in that Step (2) includes the following steps: 1) synthesizing primers for constructing crRNA transcription template DNA; the primers include a forward primer with a nucleotide sequence as shown in SEQ ID NO.12 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.13; 2) using the primers obtained in step 1) to perform PCR amplification to obtain a template DNA of crRNA; 3) Using the DNA sequence obtained in step 2) as a transcription template, in vitro transcription is performed and crRNA is purified.

9. A method for knocking down zebrafish mRNA using the CRISPR-hfCas13x editing system according to any one of claims 4 to 5, characterized in that: The zCas13x mRNA and the crRNA are microinjected into zebrafish fertilized eggs and cultured, so that the CRISPR-hfCas13x editing system can direct the shearing of the target mRNA and knock it down in the zebrafish through crRNA mediation.

10. Use of the crRNA described in any one of claims 1 to 3 and / or the CRISPR-hfCas13x editing system described in any one of claims 4 to 5 in the preparation of a kit for knocking down zebra mRNA.

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