A method for constructing an htr6 gene deletion type zebrafish epilepsy model

Abstract

This invention relates to the field of gene editing technology, and more particularly to a method for constructing a zebrafish epilepsy model with htr6 gene deletion. Using CRISPANT technology, this invention designs suitable target sites on the htr6 gene in zebrafish, and co-injects specific sgRNA synthesized in vitro with Cas9 protein into zebrafish fertilized eggs via microinjection, successfully constructing a zebrafish epilepsy model. This invention can more efficiently and accurately silence specific genes in the genome of an organism, and is simple and low-cost to produce. It can also simultaneously cleave multiple sites on the target gene, silencing any number of individual genes. The zebrafish epilepsy model provided by this invention lays a solid foundation for further research on the relationship between htr6 gene mutations and the pathogenesis of epilepsy, as well as for screening antiepileptic drugs.

Description

Technical Field

[0001] This invention relates to the field of gene editing technology, and in particular to a method for constructing an htr6 gene deletion-type zebrafish epilepsy model. Background Technology

[0002] CRISPR (clustered, regularly interspaced, short palindromic repeats) is an immune mechanism developed by bacteria to degrade invading viral DNA or other foreign DNA. In bacteria and archaea, the CRISPR system is divided into three classes. Class I and III require multiple CRISPR-related proteins (Cas proteins) to function, while Class II systems only require one Cas protein, which facilitates their widespread application. Currently, the CRISPR / Cas9 system is the most widely used. Researchers have discovered that using multiple sgRNAs to target the same gene can maximize the editing efficiency of KO (knockout) and have named this method CRISPANT technology.

[0003] Zebrafish, as vertebrates, possess a complex nervous system and a genetic structure similar to humans, sharing approximately 70% of their genes. About 84% of genes known to be associated with human diseases are also expressed in zebrafish. Compared to rodents such as mice, genetic manipulation of zebrafish is simpler, making them more suitable for establishing epilepsy models. Previous WES results from epilepsy patients identified the gene HTR6, which may cause epilepsy; however, no reports of htr6-mutated epileptic zebrafish strains have been found. Summary of the Invention

[0004] In view of this, the present invention provides a method for constructing an htr6 gene deletion-type zebrafish epilepsy model. The present invention designs a suitable target site on the htr6 gene and successfully breeds an htr6 gene deletion-type zebrafish epilepsy model.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides the application of htr6 gene knockout in the preparation of a zebrafish epilepsy model. Specifically, the knockout or knockdown is achieved using CRISPANT technology. Specifically, a 3bp sequence is inserted into exon 1 of the zebrafish htr6 gene, deleting 4bp, to breed htr6 gene-deficient zebrafish, thus creating a zebrafish epilepsy model.

[0007] This invention provides the application of sgRNA targeting exon 1 of the htr6 gene in constructing a zebrafish epilepsy model, or in screening anti-epileptic drugs.

[0008] This invention provides a combination of sgRNAs for constructing a zebrafish epilepsy model by specifically knocking out the htr6 gene in zebrafish using CRISPANT. Through CRISPANT technology, 3 bp (GCC) was inserted into exon 1 of the zebrafish htr6 gene and 4 bp (ACGG) was deleted. Finally, zebrafish with the htr6 gene deletion were bred, thus obtaining a zebrafish epilepsy model.

[0009] The present invention also provides sgRNAs targeting the zebrafish htr6 gene, including sgRNAs with sequences as shown in SEQ ID NO:1 or 2.

[0010] In some specific embodiments, the sgRNAs targeting the zebrafish htr6 gene of the present invention include sgRNA1 and sgRNA4, the sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. The two sgRNAs targeting the same gene can maximize editing efficiency and enable more effective gene editing.

[0011] The present invention also provides a reagent for knocking out the zebrafish htr6 gene, comprising Cas9 protein and the sgRNA as described in claim 2.

[0012] In some implementations, the reagent for knocking out the zebrafish htr6 gene also includes 10×Cas9 nuclease reaction buffer and nuclease-free H2O. In some specific embodiments, the reagent for knocking out the zebrafish htr6 gene comprises water and components at the following concentrations: Cas9 protein 250 ng / μl, sgRNA 100 ng / μl.

[0013] This invention also provides the application of the sgRNA or the reagents described herein in constructing a zebrafish epilepsy model.

[0014] The present invention also provides a method for constructing a zebrafish epilepsy model, wherein the reagent described in the present invention is injected into zebrafish fertilized eggs, and the zebrafish epilepsy model is obtained after hatching and screening.

[0015] In some implementations, each fertilized egg is injected with 1 nmol of the reagent described herein, wherein the final concentration of Cas9 protein in the reagent is 250 ng / μl and the final concentration of sgRNA is 100 ng / μl.

[0016] In some implementations, the incubation is carried out at a constant temperature of 28-29°C until 5 days post-flop (dpf), and then transferred to 25-28°C for 2-3 months of culture. In some specific embodiments, the incubation is carried out at a constant temperature of 28°C until 5 days post-flop (dpf), and then transferred to 25°C for 3 months of culture.

[0017] In the method for constructing a zebrafish epilepsy model provided by the present invention, the screening specifically involves: taking 3-month-old juvenile fish, cutting off the tail to extract genomic DNA for PCR amplification, performing Sanger sequencing and / or electrophoresis on the amplification products, and obtaining zebrafish with the htr6 gene deletion based on the sequencing and / or electrophoresis results.

[0018] In some embodiments, the primer sequences for the PCR amplification are shown in SEQ ID NO: 5-6.

[0019] This invention also provides the application of the zebrafish epilepsy model constructed by the method in screening antiepileptic drugs.

[0020] This invention utilizes CRISPANT technology to design suitable target sites on the htr6 gene in zebrafish. Specific sgRNA (final concentration 100 ng / μL) synthesized in vitro and Cas9 protein (final concentration 250 μg / μL) are co-injected into zebrafish cells via microinjection. After 24 hours of embryo culture, embryos are selected for genotyping analysis to identify the effectiveness of the target sites. This invention enables more efficient and precise silencing of specific genes in the genome of organisms, and is simple to prepare, low in cost, and can simultaneously cleave multiple sites on the target gene, silencing any number of individual genes. The zebrafish epilepsy model provided by this invention lays a solid foundation for further research on the relationship between htr6 mutations and the pathogenesis of epilepsy, as well as for screening antiepileptic drugs. Attached Figure Description

[0021] Figure 1 A schematic diagram of the CRISPR / Cas9 firing system;

[0022] Figure 2 This is a sequence map of the target site on the htr6 gene in the genome.

[0023] Figure 3 The image shows the electrophoretic detection of four sgRNAs (M represents DNA Marker, numbered from bottom to top as 100bp, 200bp, 300bp, 400bp, 500bp, 700bp, and 1000bp; numbers 1, 2, 3, and 4 represent sgRNA1, sgRNA2, sgRNA3, and sgRNA4, respectively).

[0024] Figure 4Electrophoresis diagrams for verifying the activity of four sgRNAs (M represents DNA Marker, from bottom to top: 100bp, 200bp, 300bp, 400bp, 500bp, 700bp, 1000bp; numbers 1, 2, and 3 represent embryos after single-target injection of sgRNA1, numbers 4, 5, and 6 represent embryos after single-target injection of sgRNA2, numbers 7, 8, and 9 represent embryos after single-target injection of sgRNA3, and numbers 10, 11, and 12 represent embryos after single-target injection of sgRNA4; WT represents wild type).

[0025] Figure 5 The knockout efficiency of four sgRNA2s after gene editing in zebrafish fertilized eggs is shown. Among them, 5-A to 5-D are the results after targeting sgRNA1 to sgRNA4, respectively.

[0026] Figure 6 Electrophoresis diagram for genotyping of the htr6F0 generation (M represents DNA Marker, from bottom to top: (100bp, 200bp, 300bp, 400bp, 500bp, 700bp, 1000bp); numbers 1-5 are the F0 generation tail cutting sequence numbers, WT represents wild type;

[0027] Figure 7 Electrophoresis diagram for genotyping of htr6F1 generation (M represents DNA Marker, from bottom to top: (100bp, 200bp, 300bp, 400bp, 500bp, 700bp, 1000bp); numbers 1-6 are the F1 generation tail cutting sequence numbers, WT is wild type);

[0028] Figure 8 Comparison of sequencing peak diagrams of wild-type and mutant htr6F1 generation;

[0029] Figure 9 The base alignment results are for htr6 homozygous mutant zebrafish and wild-type zebrafish.

[0030] Figure 10 These are representative electrophysiological signals of wild-type and homozygous mutant juvenile fish of htr6. Detailed Implementation

[0031] This invention provides a method for constructing an htr6 gene-deleted zebrafish epilepsy model. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0032] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0033] The present invention will be further illustrated below with reference to the embodiments:

[0034] Example 1

[0035] 1) Design CRISPR / Cas9 gene knockout target sites and detection primers

[0036] The genomic DNA sequence of the zebrafish htr6 gene (gene ID: 568269) was searched on the National Center for Biotechnology Information (NCBI). Based on the CRISPR / Cas9 knockout principle... Figure 1 The target site for the htr6 gene was designed on the website CHOPCHOP (http: / / chopchop.cbu.uib.no / ). The selection of the target site must follow this standard: 5'-GG-(N)18-NGG-3'. The 5' GG dinucleotide is part of the T7 promoter, and this restriction does not apply when designing the target site, but the 3' end of the target site must be NGG. The target site must be located within the gene's structural domain to ensure that the insertion or deletion of bases at the target site can affect the entire structural domain of the htr6 gene, thereby altering gene expression. The target site for the gene to be knocked out is located in exon 1 of the htr6 gene (…). Figure 2 The sgRNA sequences are shown in Table 1.

[0037] Table 1 sgRNA sequences

[0038]

[0039] Primers were designed using Primer Premier 3.0 software on a genomic region approximately 200 bp upstream and downstream of the CRISPANT target site of the htr6 gene (as shown in Table 2).

[0040] Table 2 Primer sequences

[0041]

[0042] 2) sgRNA synthesis and quality control

[0043] Using the primers designed above, a PCR experiment was performed to check for errors in the designed sgRNA sequence. After confirming that there were no errors, the designed sgRNA was sent to a commercial company (Nanjing Genscript Biotech Co., Ltd., hereinafter referred to as Nanjing Genscript) for synthesis.

[0044] The four received sgRNAs were centrifuged at 14,000 rpm for 10 min to precipitate the RNA powder. Then, 15 μL of RNase-free double-distilled water was added to dissolve the RNA powder. The quality of the dissolved sgRNAs was then assessed.

[0045] First, take 1 μL of sgRNA solution and measure its concentration using a UV spectrophotometer, recording the concentration and OD260 / 280 data for each sgRNA. Second, mix 1 μL of sgRNA solution with loading buffer and perform agarose gel electrophoresis to check if the sgRNA forms a single band. If the sgRNA concentration is high (at least above 600 ng / μL) and the electrophoretic bands are uniformly single (e.g., ...), then the sgRNA is considered a single band. Figure 3 As shown in the figure, microinjection experiments can then be performed.

[0046] Figure 3 The results showed that all four sgRNA bands were single bands, free of impurities, and of high concentration, making them suitable for microinjection experiments.

[0047] 3) Activity verification of a single sgRNA

[0048] Before formal targeting, it is necessary to test whether the designed sgRNA can effectively edit the target site. Therefore, single sgRNA activity verification was performed. Cas9 protein (Nanjing GenScript, Z03389-50) was complexed with four different sgRNAs according to the system in Table 3, resulting in a final concentration of 250 ng / μl for Cas9 protein and 100 ng / μl for sgRNA. Approximately 1 nL of the Cas9 protein and sgRNA mixture was injected into one-cell stage fertilized eggs. The injected fertilized eggs were placed in E3 water and incubated at 28°C. Embryo phenotypes were observed under a stereomicroscope, and normally developing embryos were selected for target site mutation analysis.

[0049] Table 3. SgRNA-Cas9 protein complexation system

[0050]

[0051]

[0052] 4) The effectiveness of Sanger sequencing in detecting sgRNA

[0053] After microinjection into zebrafish embryos, some normally developing early embryos were selected to test for mutations in the htr6 gene, in order to confirm in advance whether the selected target site was effective and whether the microinjection operation was standardized.

[0054] a. Extracting zebrafish genome

[0055] Twenty-four hours after fertilization (24 hpf), one tube of wild-type embryos (as control) and three tubes of embryos from the post-injection experimental group were collected into 1.5 mL Eppendorf tubes (five embryos per tube), and lysis buffer was added to extract genomic DNA.

[0056] b. PCR amplification of the target sequence

[0057] After extracting genomic DNA, primer sequences were designed using PrimerPremier 3.0 software based on a genomic region approximately 200 bp upstream and downstream of the CRISPANT target site to amplify the target DNA fragment.

[0058] Table 4 PCR reaction system

[0059]

[0060] After vortexing and mixing, centrifuge and perform amplification on a PCR instrument. The reaction conditions are: pre-denaturation at 95℃ for 5 min, followed by 35 cycles of (denaturation at 95℃ for 30 s, annealing at 56℃ for 30 s, extension at 72℃ for 30 s), and then 72℃ for 8 min. After the reaction, centrifuge the PCR product, and take 2 μL of sample, spot it onto a 1.3% agarose gel for electrophoresis to check if the PCR product size is correct. Figure 4 As shown.

[0061] Figure 4 The results showed that the band was single and could be sent to a commercial company (Sangon Biotech (Shanghai) Co., Ltd.) for Sanger sequencing.

[0062] c. If the PCR product is correct, send the PCR product for Sanger sequencing and analyze the sequencing peak chromatogram (e.g., Figure 5 (As shown) to initially obtain information on insertion or deletion, and after comparison with the Tide website to determine the sgRNA knockout efficiency, after confirming the effectiveness of sgRNA, formal injection is performed.

[0063] Table 5. Knockout efficiency of single sgRNA

[0064] sgRNA1 sgRNA2 sgRNA3 sgRNA4 Knockout efficiency 82％ 12％ 16％ 71％

[0065] 4) Microinjection of zebrafish embryos

[0066] Within 30 minutes of fertilization, the embryos are aspirated with a pipette and transferred to a microinjection culture dish made of agarose.

[0067] Prior to microinjection, Cas9 protein and two different sgRNAs (sgRNA1 and sgRNA4) were thoroughly mixed to prepare a mixture with a final concentration of 250 ng / μl for Cas9 protein and 100 ng / μl for each sgRNA. Approximately 1 nL of the Cas9 protein and sgRNA mixture was injected into one-cell stage fertilized eggs. The injected fertilized eggs were placed in E3 water and incubated at 28°C. Embryo phenotypes were observed under a stereomicroscope, and normally developing embryos were selected for target site mutation analysis.

[0068] Three tubes of embryos (5 embryos per tube) were selected to test the knockout efficiency. The testing steps were the same as above. After confirming that the knockout was effective, the remaining embryos were cultured to 3 months of age.

[0069] 5) Screening of F0 generation mutant zebrafish

[0070] a. Extracting zebrafish genome

[0071] After the embryos reached 3 months of age, some tail fin tissue of adult zebrafish was collected in a 1.5 mL centrifuge tube, and lysis buffer was added to the EP tube to extract genomic DNA.

[0072] b. PCR amplification of the target sequence

[0073] After extracting genomic DNA, the target DNA fragment was amplified using the primer sequences in Table 2 and the PCR reaction system in Table 4. The reaction conditions were: pre-denaturation at 95℃ for 5 min, followed by 35 cycles of denaturation at 95℃ for 30 s, annealing at 56℃ for 30 s, and extension at 72℃ for 30 s, and then 72℃ for 8 min. After the reaction, the PCR products were centrifuged, and 2 μL of the sample was loaded onto a 1.3% agarose gel for electrophoresis to check the size of the PCR products.

[0074] c. If the PCR product is correct, send the PCR product for Sanger sequencing. The insertion or deletion information is obtained from the sequencing peak chromatogram. Screen for F0 generation zebrafish carrying the mutation. Experimental results are as follows: Figure 6 As shown.

[0075] Figure 6 The results showed that the target band was single, and the sample was sent to a commercial company (Sangon Biotech (Shanghai) Co., Ltd.) for Sanger sequencing.

[0076] 6) Obtain the F1 generation of heritable zebrafish mutants.

[0077] The F0 generation of zebrafish mutants was identified through a series of screenings. Then, the F0 generation mutants were crossed with wild-type zebrafish to obtain F1 generation embryos, which were cultured at 28°C. The survival rate of the F1 generation was observed in the early stage.

[0078] If a mutation is detected in the F1 generation embryos, the F1 generation of zebrafish mutants is raised to 2-3 months of age. Then, the tails of each adult F1 generation zebrafish are clipped to screen for F1 generation mutants (the specific method is described in 5). The experimental results are as follows: Figure 7 As shown.

[0079] Figure 7 The results showed that the target band was single, and the sample was sent to a commercial company (Sangon Biotech (Shanghai) Co., Ltd.) for Sanger sequencing.

[0080] Analysis of the Sanger sequencing results of the selected F1 generation mutants revealed that the htr6 gene contains a 3bp insertion and a 4bp deletion in exon 1. Experimental results are as follows... Figures 8-9 As shown, the part within the blue box represents the missing 4bp sequence: ACGG.

[0081] according to Figure 8 Sequencing peak diagrams show that, compared to the wild-type sequence, the mutant sgRNA sequence contains a 3bp insertion and a 4bp deletion, resulting in a frameshift mutation. Note: Figure 8 The sequencing results were obtained using primer htr6-F.

[0082] 7) F2 representative experiment

[0083] The F1 generation, identified through a series of screenings, carried the mutation (3bp insertion and 4bp deletion) and was raised to sexual maturity. Immediately afterwards, F1 embryos with the same mutation were self-crossed to obtain F2 embryos, which were then cultured at 28°C. Fifty juveniles that reached 5 days post-flop (dpf) were randomly selected for observation of the neurophysiological phenotype of the F2 mutant juveniles. Neurophysiological testing results showed epileptiform signals in homozygous mutant juveniles. The experimental results are as follows: Figure 10 As shown.

[0084] The results showed that the deletion of the htr6 gene caused epileptic seizures in zebrafish, and the present invention successfully constructed a zebrafish epilepsy model with the deletion of the htr6 gene.

[0085] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of htr6 gene knockout in the preparation of zebrafish epilepsy model.

2. Application of sgRNA targeting exon 1 of the htr6 gene in constructing a zebrafish epilepsy model with htr6 gene knockout.

3. The application according to claim 2, characterized in that, The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 1 and / or SEQ ID NO:

2.

4. Application of reagents for knocking out the htr6 gene in zebrafish in constructing a zebrafish epilepsy model.

5. The application according to claim 4, characterized in that, The reagents include sgRNA and Cas9 protein; The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 1 and / or SEQ ID NO:

2.

6. A method for constructing a zebrafish epilepsy model with htr6 gene deletion, characterized in that, A reagent that knocks out the htr6 gene in zebrafish was injected into zebrafish fertilized eggs. After hatching and screening, a zebrafish epilepsy model was obtained.

7. The construction method according to claim 6, characterized in that, The reagents used to knock out the zebrafish htr6 gene include sgRNA and Cas9 protein; The nucleotide sequence of the sgRNA is shown in SEQ ID NO: 1 and / or SEQ ID NO:

2.

8. The construction method according to claim 6 or 7, characterized in that, Each fertilized egg was injected with 1 nl of the reagent, wherein the final concentration of Cas9 protein in the reagent was 250 ng / μl and the final concentration of sgRNA was 100 ng / μl.

9. The construction method according to claim 6, characterized in that, The incubation process involves: constant temperature culture at 28~29℃ until 5 days post-fertilization (dpf), followed by transfer to 25~28℃ for culture until 2~3 months of age.

10. The construction method according to claim 6, characterized in that, The screening process includes: taking 2-month-old juvenile fish, cutting off the tail to extract genomic DNA for PCR amplification, performing Sanger sequencing and / or electrophoresis on the amplification products, and obtaining zebrafish with the htr6 gene deletion based on the sequencing and / or electrophoresis results.

11. The construction method according to claim 10, characterized in that, The primer sequences for the PCR amplification are shown in SEQ ID NO: 5~6.

12. The application of the zebrafish epilepsy model obtained by the construction method according to any one of claims 6 to 11 in screening antiepileptic drugs.

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