SgRNA combination and application thereof in construction of zebrafish srsf5b gene mutant model

Abstract

The invention belongs to the technical field of gene knockout, and particularly relates to an sgRNA combination and application thereof in construction of a zebrafish srsf5b gene mutant model. The sgRNA combination provided by the invention comprises sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16, and the sgRNA combination sequentially comprises nucleotide sequences as shown in SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 13 and SEQ ID NO: 16. According to the sgRNA combination provided by the invention, the srsf5b gene of the zebrafish can be specifically knocked out, and the srsf5b gene mutant zebrafish is bred.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene knockout, and particularly relates to an sgRNA combination and an application thereof in constructing a zebrafish srsf5b gene mutant model. Background Art

[0002] The serine / arginine-rich (SR) splicing factor family plays an important role in the activation, repression, export, stabilization, and translation of mRNA splicing. srsf5 (serine- and arginine-rich splicing factor 5) is a gene encoding a protein that is a member of the serine / arginine-rich (SR) pre-mRNA splicing factor family. It forms part of the spliceosome and contains an RNA recognition motif (RRM) for RNA binding and an RS domain for binding other proteins. The RS domain is rich in serine and arginine residues and promotes interactions between different SR splicing factors. In addition to being essential for mRNA splicing, SR proteins have been shown to be involved in mRNA export from the nucleus and translation. SR splicing factor 5 (SRSF5) is a glucose-induced protein that has been shown to promote cancer development and progression by regulating the splicing of multiple genes.

[0003] SRSF5 has been shown to promote aerobic glycolysis in colorectal cancer cells by increasing glucose consumption and lactate production. In addition, a preliminary study showed that srsf5 gene knockdown could inhibit glycolysis and cell proliferation by reducing the expression of PKM2 in non-small cell lung cancer cells, but the detailed mechanism is still unclear. Some scholars have used srsf5 knockout of Exon3 to Exon6 to - / - Mutant mice have shown that alternative splicing regulated by srsf5 plays a key role in heart development. Currently, research on the physiological functions and mechanisms of action of the srsf5 gene in fish tissues and organs is relatively scarce, and no studies have yet examined the effects of srsf5b gene deletion on fish tissues and organs. It is particularly important to examine the impact of the srsf5b gene on the normal physiological functions of zebrafish tissues and organs by constructing mutant models.

[0004] At present, the main research method for the role of gene function in zebrafish is the gene knockdown method mediated by morpholino-modified antisense oligonucleotide technology (Morpholino). Although this technical method can quickly study the function of the target gene, it also has disadvantages: 1. The knockdown effect may vary greatly between different cell types and different target genes. 2. There may be side effects that non-specifically affect other non-target RNAs or proteins. 3. When gene expression is reduced at the mRNA level, the gene expression product still plays a role, and some special phenotypes are difficult to observe. 4. It can only reduce the expression of the target gene in the zebrafish in the short term, and it is impossible to achieve long-term observation of functional disorders caused by the loss of gene expression. 5. Morpholino may have toxic effects on cells or organisms in some cases. High concentrations or long-term exposure to Morpholino may cause adverse cellular reactions or affect embryonic development. Summary of the Invention

[0005] The purpose of the present invention is to provide a sgRNA combination and its application in constructing a zebrafish srsf5b gene mutant model, specifically knocking out the zebrafish srsf5b gene, and breeding srsf5b gene mutant zebrafish.

[0006] The present invention provides an sgRNA combination, comprising sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16;

[0007] The sgRNA2 includes the nucleotide sequence shown in SEQ ID NO: 2;

[0008] The sgRNA5 includes the nucleotide sequence shown in SEQ ID NO: 5;

[0009] The sgRNA6 includes the nucleotide sequence shown in SEQ ID NO: 6;

[0010] The sgRNA8 includes the nucleotide sequence shown in SEQ ID NO: 8;

[0011] The sgRNA13 includes the nucleotide sequence shown in SEQ ID NO:13;

[0012] The sgRNA16 includes the nucleotide sequence shown in SEQ ID NO:16.

[0013] The present invention also provides a primer combination for synthesizing the sgRNA combination described in the above technical solution, wherein the primer combination includes an upstream primer combination and a downstream primer R-Common;

[0014] The upstream primer combination includes sgRNA2-F, sgRNA5-F, sgRNA6-F, sgRNA8-F, sgRNA13-F and sgRNA16-F;

[0015] The sgRNA2-F includes the nucleotide sequence shown in SEQ ID NO: 21;

[0016] The sgRNA5-F includes the nucleotide sequence shown in SEQ ID NO: 24;

[0017] The sgRNA6-F includes the nucleotide sequence shown in SEQ ID NO: 25;

[0018] The sgRNA8-F includes the nucleotide sequence shown in SEQ ID NO: 27;

[0019] The sgRNA13-F includes the nucleotide sequence shown in SEQ ID NO: 32;

[0020] The sgRNA16-F includes the nucleotide sequence shown in SEQ ID NO: 35;

[0021] The downstream primer R-Common includes the nucleotide sequence shown in SEQ ID NO:36.

[0022] The present invention also provides a method for preparing the sgRNA combination described in the above technical solution, comprising the following steps:

[0023] The pYSY-sgRNA plasmid was amplified by PCR using sgRNA2-F, sgRNA5-F, sgRNA6-F, sgRNA8-F, sgRNA13-F and sgRNA16-F in the primer combination described in the above technical solution, respectively, and R-Common. After obtaining the amplified products, they were transcribed in vitro to obtain sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16.

[0024] The present invention also provides the use of the sgRNA combination described in the above technical solution, the primer combination described in the above technical solution, or the sgRNA combination obtained by the preparation method described in the above technical solution in knocking out the zebrafish srsf5b gene and / or constructing a zebrafish srsf5b gene mutant model.

[0025] The present invention also provides a kit for knocking out the zebrafish srsf5b gene and / or constructing a zebrafish srsf5b gene mutant model, comprising the sgRNA combination described in the above technical solution or the primer combination described in the above technical solution or the sgRNA combination obtained by the preparation method described in the above technical solution.

[0026] Preferably, the kit further comprises Cas9 protein.

[0027] The present invention also provides a method for constructing a zebrafish srsf5b gene mutant model, comprising the following steps:

[0028] The mixture of sgRNA combination and Cas9 protein is introduced into zebrafish fertilized eggs, and embryos with effective knockout are selected and cultured to adult fish to obtain F0 generation mutant zebrafish; the sgRNA combination described in the above technical solution is the sgRNA combination or the sgRNA combination synthesized using the primer combination described in the above technical solution or the sgRNA combination prepared by the preparation method described in the above technical solution

[0029] The F0 generation mutant zebrafish is hybridized with wild-type zebrafish to obtain F1 generation embryos, the F1 generation embryos are screened for mutant embryos, and the embryos are cultured to adult fish to obtain a zebrafish srsf5b gene mutant model.

[0030] Preferably, the concentration of the sgRNA combination in the mixture of sgRNA combination and Cas9 protein is 200 ng / μL, and the concentration of Cas9 protein is 400 ng / μL.

[0031] Preferably, the mass ratio of sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16 in the sgRNA combination is 1:1:1:1:1:1.

[0032] Preferably, the method for selecting an effective knockout embryo comprises the following steps:

[0033] Fertilized eggs developed to 24 hpf were taken and genomic DNA was prepared;

[0034] Using the genomic DNA as a template, PCR amplification is performed using upstream amplification primers and downstream amplification primers to obtain a PCR amplification product;

[0035] The PCR amplification product is compared with the wild-type zebrafish srsf5b gene. If the comparison results are different, the fertilized egg that develops to 24 hpf is an effective knockout embryo;

[0036] The upstream amplification primer includes the nucleotide sequence shown in SEQ ID NO: 17;

[0037] The downstream amplification primer includes the nucleotide sequence shown in SEQ ID NO: 18.

[0038] Beneficial effects:

[0039] The present invention provides an sgRNA combination, comprising sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16; the sgRNA2 comprises the nucleotide sequence shown in SEQ ID NO: 2; the sgRNA5 comprises the nucleotide sequence shown in SEQ ID NO: 5; the sgRNA6 comprises the nucleotide sequence shown in SEQ ID NO: 6; the sgRNA8 comprises the nucleotide sequence shown in SEQ ID NO: 8; the sgRNA13 comprises the nucleotide sequence shown in SEQ ID NO: 13; and the sgRNA16 comprises the nucleotide sequence shown in SEQ ID NO: 16. The present invention uses Crispant technology to design multiple sgRNAs with the second exon of the zebrafish srsf5b gene as the target, and simultaneously targets the zebrafish srsf5b gene, which can maximize the editing efficiency and more effectively perform gene editing on the zebrafish srsf5b gene. Multiple nucleic acid sequences are inserted or deleted in the zebrafish srsf5b gene, specifically knocking out zebrafish. Each individual produced has cells of different genotypes, and srsf5b gene mutant zebrafish are bred. This helps to reveal the function of the zebrafish srsf5b gene, lays the foundation for developing srsf5b gene mutation-type diseases, and explores the regulatory relationship between SR protein family members and their target gene alternative splicing during development. In addition, the DNA site after gene knockout using the sgRNA combination of the present invention can be stably inherited in zebrafish, phenotypic observation is not limited by time, and gene screening can be performed quickly and in large quantities. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0041] Figure 1 This is the result of agarose gel electrophoresis identification of sgRNA synthesized in vitro;

[0042] Figure 2 This is the result of sequencing and identifying the target genomic DNA cut by sgRNA in step 2 of Example 2;

[0043] Figure 3 This is a diagram showing the sequencing results of the target genomic DNA cut by sgRNA II in step 2 of Example 2;

[0044] Figure 4 This is the result of sequencing and identifying the target genomic DNA by sgRNA triple-cutting in step 2 of Example 2;

[0045] Figure 5 This is the result of sequencing and identifying the target genomic DNA by sgRNA four-cutting in step 2 of Example 2;

[0046] Figure 6 This is the sequencing peak diagram of F1 zebrafish carrying the -1+32bp deletion mutation;

[0047] Figure 7 This is the sequencing peak diagram of F1 zebrafish carrying a -1+6bp frameshift mutation;

[0048] Figure 8 This is the sequencing peak diagram of F1 zebrafish carrying a -3bp frameshift mutation;

[0049] Figure 9 This is the sequencing peak diagram of F1 zebrafish carrying a -1bp frameshift mutation. DETAILED DESCRIPTION

[0050] The present invention provides an sgRNA combination, comprising sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16;

[0051] The sgRNA2 includes the nucleotide sequence shown in SEQ ID NO: 2;

[0052] The sgRNA5 includes the nucleotide sequence shown in SEQ ID NO: 5;

[0053] The sgRNA6 includes the nucleotide sequence shown in SEQ ID NO: 6;

[0054] The sgRNA8 includes the nucleotide sequence shown in SEQ ID NO: 8;

[0055] The sgRNA13 includes the nucleotide sequence shown in SEQ ID NO:13;

[0056] The sgRNA16 includes the nucleotide sequence shown in SEQ ID NO:16.

[0057] The present invention also provides a primer combination for synthesizing the sgRNA combination described in the above technical solution, wherein the primer combination includes an upstream primer combination and a downstream primer R-Common;

[0058] The upstream primer combination includes sgRNA2-F, sgRNA5-F, sgRNA6-F, sgRNA8-F, sgRNA13-F and sgRNA16-F;

[0059] The sgRNA2-F includes the nucleotide sequence shown in SEQ ID NO: 21;

[0060] The sgRNA5-F includes the nucleotide sequence shown in SEQ ID NO: 24;

[0061] The sgRNA6-F includes the nucleotide sequence shown in SEQ ID NO: 25;

[0062] The sgRNA8-F includes the nucleotide sequence shown in SEQ ID NO: 27;

[0063] The sgRNA13-F includes the nucleotide sequence shown in SEQ ID NO: 32;

[0064] The sgRNA16-F includes the nucleotide sequence shown in SEQ ID NO: 35;

[0065] The downstream primer R-Common includes the nucleotide sequence shown in SEQ ID NO:36.

[0066] The present invention also provides a method for preparing the sgRNA combination described in the above technical solution, comprising the following steps:

[0067] The pYSY-sgRNA plasmid was amplified by PCR using sgRNA2-F, sgRNA5-F, sgRNA6-F, sgRNA8-F, sgRNA13-F and sgRNA16-F in the primer combination described in the above technical solution, respectively, and R-Common. After obtaining the amplified products, they were transcribed in vitro to obtain sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16.

[0068] In one embodiment, the PCR amplification system described herein comprises: 40 μL 2× Mastermix, 35 μL water, 2 μL 5 μM upstream primer, 2 μL 5 μM downstream primer R-Common, and 1 μL 10 ng / μL pYSY-sgRNA plasmid. In one embodiment, the PCR amplification protocol described herein comprises a preliminary denaturation at 95°C for 3 minutes, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, and extension at 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. The specific steps of the in vitro transcription described herein are not strictly required; conventional methods in the art can be employed.

[0069] The present invention also provides the use of the sgRNA combination described in the above technical solution, the primer combination described in the above technical solution, or the sgRNA combination obtained by the preparation method described in the above technical solution in knocking out the zebrafish srsf5b gene and / or constructing a zebrafish srsf5b gene mutant model. As an embodiment, the knocking out of the zebrafish srsf5b gene described in the present invention is to knock out exon 2 of the zebrafish srsf5b gene. As an embodiment, the constructing of the zebrafish srsf5b gene mutant model described in the present invention is to construct a zebrafish srsf5b gene exon 2 mutant model.

[0070] The present invention designs an sgRNA combination with the second exon of the zebrafish srsf5b gene as the target, performs gene editing on the zebrafish srsf5b gene, can specifically knock out the zebrafish srsf5b gene, and breed srsf5b gene mutant zebrafish.

[0071] The present invention also provides a kit for knocking out the zebrafish srsf5b gene and / or constructing a zebrafish srsf5b gene mutant model, comprising the sgRNA combination described in the above technical solution or the primer combination described in the above technical solution or the sgRNA combination obtained by the preparation method described in the above technical solution.

[0072] As an embodiment, the kit of the present invention further comprises Cas9 protein.

[0073] The present invention also provides a method for constructing a zebrafish srsf5b gene mutant model, comprising the following steps:

[0074] The mixture of sgRNA combination and Cas9 protein is introduced into zebrafish fertilized eggs, and embryos with effective knockout are selected and cultured to adult fish to obtain F0 generation mutant zebrafish; the sgRNA combination described in the above technical solution is the sgRNA combination or the sgRNA combination synthesized using the primer combination described in the above technical solution or the sgRNA combination prepared by the preparation method described in the above technical solution

[0075] The F0 generation mutant zebrafish is hybridized with wild-type zebrafish to obtain F1 generation embryos, the F1 generation embryos are screened for mutant embryos, and the embryos are cultured to adult fish to obtain a zebrafish srsf5b gene mutant model.

[0076] The present invention introduces a mixture of sgRNA combination and Cas9 protein into zebrafish fertilized eggs, selects effectively knocked-out embryos, and cultured them to adult fish to obtain F0 generation mutant zebrafish.

[0077] In one embodiment, the method of introduction described in the present invention includes injection. In one embodiment, the concentration of the sgRNA combination in the mixture of the sgRNA combination and Cas9 protein described in the present invention is 200 ng / μL. In one embodiment, the concentration of the Cas9 protein in the mixture of the sgRNA combination and Cas9 protein described in the present invention is 400 ng / μL. In another embodiment, the mass ratio of sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13, and sgRNA16 described in the present invention is 1:1:1:1:1:1.

[0078] As an embodiment, the method for selecting an effectively knocked-out embryo described in the present invention includes the following steps: taking a fertilized egg that has developed to 24 hpf and preparing genomic DNA; using the genomic DNA as a template, PCR amplification is performed using an upstream amplification primer and a downstream amplification primer to obtain a PCR amplification product; comparing the PCR amplification product with the wild-type zebrafish srsf5b gene. If the comparison results are different, the fertilized egg that has developed to 24 hpf is an effectively knocked-out embryo; the upstream amplification primer includes the nucleotide sequence shown in SEQ ID NO: 17; the downstream amplification primer includes the nucleotide sequence shown in SEQ ID NO: 18.

[0079] As an embodiment, the present invention uses a zebrafish genotype identification kit to prepare the genomic DNA. As an embodiment, the present invention mixes the fertilized eggs that have developed to 24hpf with the buffer in the zebrafish genotype identification kit, reacts, and obtains the genomic DNA. As an embodiment, the reaction conditions of the present invention are 65°C for 30min, 95°C for 5min, 16°C for 1min, and storage at 4°C. As an embodiment, the ratio of the fertilized eggs that have developed to 24hpf to the buffer of the present invention is 2 pieces: 20μl. The zebrafish genotype identification kit of the present invention is preferably purchased from Nanjing Yaoshunyu Biotechnology Co., Ltd.

[0080] In one embodiment, the PCR amplification system described herein comprises, in a 30 μL volume, 1.5 μL 2× Mastermix, 2 μL 5 μM upstream primer, 2 μL 5 μM downstream primer, 2 μL genomic DNA, and the balance water. In one embodiment, the PCR amplification program described herein is as follows: initial denaturation at 96°C for 3 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s; and final extension at 72°C for 10 min.

[0081] After obtaining the F0 generation mutant zebrafish, the present invention hybridizes the F0 generation mutant zebrafish with wild-type zebrafish to obtain F1 generation embryos, screens the F1 generation embryos for mutant embryos, and cultured them to adult fish to obtain a zebrafish srsf5b gene mutant model.

[0082] As an embodiment, the wild-type zebrafish of the present invention is a wild-type AB strain zebrafish. As an embodiment, the method of screening the F1 generation embryos for embryos with mutations is the same as the method of screening embryos with effective knockouts, which will not be described in detail here.

[0083] To further illustrate the present invention, an sgRNA combination provided by the present invention and its application in constructing a zebrafish srsf5b gene mutant model are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0084] Example 1

[0085] Step 1: Crispant strategy knockdown target site design and PCR detection primers

[0086] The genomic DNA sequence of the zebrafish srsf5b gene (GeneID: 436883) was searched in the NCBI database. Functional domains were analyzed and target sites were designed. The target site for gene knockout was determined to be located in exon 2 of the zebrafish srsf5b gene. PCR amplification (sequencing) primers and sgRNA sequences were designed based on exon 2 of the zebrafish srsf5b gene. Specific sequence information is listed in Table 1.

[0087] Table 1 sgRNA and primer sequences designed based on exon 2

[0088]

[0089]

[0090] Step 2: Confirm the genotype of the target gene

[0091] (1) Preparation of genomic DNA template

[0092] Place a 24 hpf wild-type zebrafish embryo in a 200 μL PCR tube, aspirate the water, and add 10 μL of YSY buffer produced by Nanjing Yaoshunyu Biotechnology Co., Ltd. Genomic DNA template was prepared using the Zebrafish Genotyping Kit from Nanjing Yaoshunyu Biotechnology Co., Ltd. The specific reaction conditions were: 65°C for 30 min, 95°C for 5 min, 16°C for 1 min, and 4°C.

[0093] (2) PCR amplification and detection

[0094] PCR reaction system (30 μL): 15 μL 2× Mastermix (Novozyme), 11 μL ultrapure water, 1 μL amplification upstream primer (5 μM), 1 μL amplification downstream primer (5 μM) and 2 μL genomic DNA template obtained in step (1);

[0095] PCR reaction conditions: pre-denaturation at 96°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 10 min, and storage at 4°C.

[0096] 2 μL of the PCR amplified product was subjected to agarose gel electrophoresis (1%), and the PCR amplified product was sent to a commercial company for sequencing and verification.

[0097] Step 3: sgRNA in vitro synthesis and quality control

[0098] (1) In vitro synthesis of primers for sgRNA expression constructs

[0099] Design sgRNA according to Table 1, and design the forward primer for synthesizing sgRNA. The specific sequences are shown in Table 2.

[0100] Table 2 Forward primer information for synthesizing sgRNA

[0101]

[0102] Note: The bold part is the T7 promoter part, and the lowercase letters are part of the sgRNA backbone template sequence.

[0103] The reverse primers for synthesizing sgRNA1 to sgRNA16 were all universal primers R-Common, and the specific nucleotide sequence was: 5'-AAAAAAAGCACCGACTCGGTGCCAC-3' (SEQ ID NO: 36).

[0104] (2) PCR amplification and synthesis of sgRNA

[0105] PCR reaction system: 40 μL 2× Mastermix (Novozymes), 35 μL ultrapure water, 2 μL forward primer (sgRNAn-F (5 μM), 2 μL R-Common) primer (5 μM) and 1 μL pYSY-sgRNA (Nanjing Yaoshunyu Biotechnology Co., Ltd.) plasmid (10 ng / μL); among them, sgRNAn-F is any one of sgRNA1-F to sgRNA16-F, and a total of 16 PCR reaction systems are configured.

[0106] PCR reaction conditions: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; extension at 72°C for 10 min; and storage at 4°C.

[0107] The expected PCR amplification products (i.e., the obtained sgRNA in vitro transcription template sequences) are shown in Table 3.

[0108] Table 3 sgRNA in vitro transcription template sequences

[0109]

[0110]

[0111] (3) PCR product purification (nuclease-free treatment)

[0112] The PCR products obtained in step (2) were collected using a nuclease-free PCR clean up kit (purchased from Axygen), and the recovery solvent was ultrapure water free of nuclease contamination.

[0113] (4) In vitro transcription of sgRNA

[0114] The PCR products purified in step (3) were transcribed in vitro using T7 RNA polymerase. Using an RNA in vitro transcription kit (MAXIscript T7, Ambion, USA), 4 μL of 10× Transcription Buffer, 2 μL of 10 mM ATP, 2 μL of 10 mM CTP, 2 μL of 10 mM GTP, 2 μL of 10 mM UTP, 4 μL of T7 RNA polymerase mix, and 24 μL of the PCR product template DNA purified in step (3) were added in sequence according to the kit instructions. After gently flicking and centrifuging, the mixture was incubated in a 37°C water bath for 3 h. 1.5 μL of DNase I (Ambion, USA) was added and the mixture was incubated in a 37°C water bath for 15 min to remove the template.

[0115] 160 μL DEPC water was then added to expand the volume to 200 μL. 20 μL of nuclease-free 3M sodium acetate (pH 5.2) and 3 volumes of anhydrous ethanol (Sanggong) were also added, and precipitation was allowed to proceed at -80°C overnight. The mixture was centrifuged at 12,000 g for 20 min at 4°C. After removing the supernatant, nuclease-free 75% ethanol was added and the mixture was centrifuged at 12,000 g for 20 min at 4°C. After removing the supernatant, the precipitate was dried in a fume hood and then resuspended in 20 μL of nuclease-free ultrapure water to obtain sgRNAs 1 to 16, which were stored in a -80°C refrigerator for later use.

[0116] (5) sgRNA quality identification

[0117] The sgRNA1, sgRNA3, sgRNA9 and sgRNA16 obtained in step (4) are mixed in equal amounts at a ratio of 1:1:1:1 to obtain sgRNA one; the sgRNA2, sgRNA5, sgRNA10 and sgRNA15 obtained in step (4) are mixed at a mass ratio of 1:1:1:1 to obtain sgRNA two; the sgRNA4, sgRNA7, sgRNA11 and sgRNA14 obtained in step (4) are mixed at a mass ratio of 1:1:1:1 to obtain sgRNA three; the sgRNA6, sgRNA8, sgRNA12 and sgRNA13 obtained in step (4) are mixed at a mass ratio of 1:1:1:1 to obtain sgRNA four;

[0118] Take 1 μL of sgRNA 1 to 4 and perform agarose gel electrophoresis (1%) to identify their integrity. Figure 1 As shown. Among them, Figure 1 From left to right, lane 1 is DNA Marker, and from bottom to top, they are 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp, 3000bp, and 5000bp; lanes 2 to 5 are sgRNA 1, sgRNA 2, sgRNA 3, and sgRNA 4. Figure 1 The sgRNA concentrations were also measured, with sgRNA 1 at 1023 ng / μl, sgRNA 2 at 1125 ng / μl, sgRNA 3 at 1067 ng / μl, and sgRNA 4 at 997 ng / μl.

[0119] Example 2

[0120] Step 1: Microinjection of zebrafish fertilized eggs

[0121] Zebrafish fertilized eggs were collected according to conventional methods, and sgRNA 1, sgRNA 2, sgRNA 3, and sgRNA 4 obtained in Example 1 were mixed at equal concentrations to obtain an sgRNA combination; the sgRNA combination (final concentration of 200 ng / μL) and Cas9 protein (final concentration of 400 ng / μL) were mixed and microinjected into zebrafish fertilized eggs at an injection volume of 1 nL / embryo.

[0122] Step 2: Confirmation of the efficiency of sgRNA-guided Cas9 targeting and cutting the target genomic DNA sequence

[0123] (1) Preparation of target genomic DNA template

[0124] When the embryos injected in step 1 developed to 24 hpf, 16 single embryos were randomly selected and genomic DNA was prepared using a zebrafish genotyping kit (purchased from Nanjing Yaoshunyu Biotechnology Co., Ltd.); the specific reaction conditions were: 65°C for 30 min, 95°C for 5 min, 16°C for 1 min, and stored at 4°C.

[0125] (2) PCR detection

[0126] PCR amplification was performed using the primer pair srsf5b-E2-F1 / R1 in Table 1 and the genomic DNA obtained in step (1) as a template. The specific reaction system and reaction procedure are as follows:

[0127] PCR reaction system (30 μL): 15 μL 2× Mastermix, 11 μL ultrapure water, 1 μL srsf5b-E2-F1 (5 μM), 1 μL srsf5b-E2-R1 (5 μM) and 2 μL genomic DNA template.

[0128] The PCR reaction conditions were as follows: pre-denaturation at 96°C for 3 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for 35 cycles; final extension at 72°C for 10 min, and storage at 4°C.

[0129] (3) Detection and sequencing of PCR products

[0130] After the PCR products obtained in step (2) were electrophoresed to confirm the band size, all positive amplification products were sent to a commercial company for sequencing. The sequencing primers were srsf5b-E2-F / R, and the results were obtained by direct reading of the map. Figures 2 to 5 As shown, the red box indicates the Indel mutation near CRISPR. sgRNA group (1, 3, 9, 16), the Indel mutation near CRISPR16 in the effective sequencing ( Figure 2 ). Two groups of sgRNA (2, 5, 10, 15) have Indel mutations near CRISPR2 and Indel mutations near CRISPR5 in effective sequencing. Figure 3 ). In the three sgRNA groups (4, 7, 11, and 14), no Indel mutations were found in the effective sequencing results ( Figure 4Four sgRNA groups (6, 8, 12, and 13) showed effective sequencing results, including indel mutations near CRISPR6, CRISPR8, and CRISPR13. Therefore, using zebrafish embryos as a reactor, sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13, and sgRNA16 effectively edited the zebrafish srsf5b gene. These highly active sgRNAs were mixed and injected for subsequent Crispant knockdown experiments.

[0131] Step 3: Screening and identification of F1 mutants knocking down the zebrafish srsf5b gene using the Crispant strategy

[0132] (1) Preparation of candidate F1 mutant genomic DNA template

[0133] a. Raise the F0 embryos injected with Cas9 and sgRNA in Step 2 to sexual maturity and then mate with wild-type AB zebrafish to obtain F1 embryos. Raise the F1 embryos to sexual maturity as per standard procedures, perform genotyping, and screen for genome-edited mutants.

[0134] b. Tissue sampling

[0135] Forty three-month-old F1 adult fish were collected, and part of the tail fin tissue was cut off. The tissues were placed in 200 μL PCR tubes in order and then stored on ice.

[0136] c. Genomic DNA template preparation

[0137] Add 10 μL of YSY buffer produced by Nanjing Yaoshunyu Biotechnology Co., Ltd. to the 200 μL PCR tube containing the tail fin tissue. After rapid centrifugation, place the PCR tube in a PCR instrument and perform the following reaction: 65°C for 30 min, 95°C for 5 min, 16°C for 1 min, and 4°C for 1 min.

[0138] (2) PCR amplification of genomic DNA fragments

[0139] PCR amplification reaction system (20 μL): 10 μL 2× Mastermix (Novozymes), 7 μL ultrapure water, 1 μL srsf5b-E2-F1 (5 μM), 1 μL srsf5b-E2-R1 (5 μM), and 1 μL genomic DNA template;

[0140] PCR amplification conditions: pre-denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 30 s; extension at 72°C for 10 min; and storage at 4°C.

[0141] (3) Direct sequencing of PCR products

[0142] The obtained PCR products were sent to a commercial company for direct Sanger sequencing. The sequencing primers were srsf5b-E2-F1. As a result, four zebrafish (F1) with heterozygous srsf5b frameshift mutations were screened. The numbers and mutation status of the four mutant zebrafish are shown in Table 4. The specific sequence sequencing results are shown in Table 4. Figures 6 to 9 and Table 5.

[0143] Table 4 Mutation types of mutants

[0144]

[0145]

[0146] Table 5 Partial genome sequence and predicted protein sequence

[0147]

[0148]

[0149] Note: The bold part is the base sequence deleted in the mutant compared to the wild type, and the underlined part () is the base sequence inserted in the mutant compared to the wild type.

[0150] according to Figures 6 to 9 As can be seen from Table 5, the F1 generation zebrafish numbered 1, 3, 4, and 8 have mutations in exon 2 compared to the wild type. Zebrafish number 1 has a -1bp+32bp frameshift heterozygous deletion mutation starting at 209bp. Figure 6 ) It can be judged that the sequencing result of No. 1 is a -1bp+32bp frameshift heterozygous mutant. Zebrafish No. 3 begins to show a -1+6bp frameshift heterozygous deletion mutation at 167bp. Peak diagram ( Figure 7 ) It can be judged that the sequencing result of No. 3 is a -1+6bp frameshift heterozygous mutant. Zebrafish No. 4 began to show a -3bp frameshift heterozygous deletion mutation at 168bp. The peak diagram ( Figure 8 ) It can be judged that the sequencing result of No. 4 is a -3bp frameshift heterozygous mutant; the No. 8 zebrafish began to show a -1bp frameshift heterozygous deletion mutation at 172bp, and the peak diagram ( Figure 9 ) It can be determined that the sequencing result of No. 8 is a -1bp frameshift heterozygous mutant. The method provided by the present invention can construct a zebrafish model with srsf5b gene mutation.

[0151] Based on the above content, it can be seen that the sgRNA combination provided by the present invention can specifically knock out the zebrafish srsf5b gene and breed srsf5b gene mutant zebrafish.

[0152] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An sgRNA combination, characterized in that The sgRNA combination includes sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16; The sgRNA2 includes the nucleotide sequence shown in SEQ ID NO: 2; The sgRNA5 includes the nucleotide sequence shown in SEQ ID NO: 5; The sgRNA6 includes the nucleotide sequence shown in SEQ ID NO: 6; The sgRNA8 includes the nucleotide sequence shown in SEQ ID NO: 8; The sgRNA13 includes the nucleotide sequence shown in SEQ ID NO:13; The sgRNA16 includes the nucleotide sequence shown in SEQ ID NO:

16.

2. A primer combination for synthesizing the sgRNA combination according to claim 1, characterized in that: The primer combination includes an upstream primer combination and a downstream primer R-Common; The upstream primer combination includes sgRNA2-F, sgRNA5-F, sgRNA6-F, sgRNA8-F, sgRNA13-F and sgRNA16-F; The sgRNA2-F includes the nucleotide sequence shown in SEQ ID NO: 21; The sgRNA5-F includes the nucleotide sequence shown in SEQ ID NO: 24; The sgRNA6-F includes the nucleotide sequence shown in SEQ ID NO: 25; The sgRNA8-F includes the nucleotide sequence shown in SEQ ID NO: 27; The sgRNA13-F includes the nucleotide sequence shown in SEQ ID NO: 32; The sgRNA16-F includes the nucleotide sequence shown in SEQ ID NO: 35; The downstream primer R-Common includes the nucleotide sequence shown in SEQ ID NO:

36.

3. The method for preparing the sgRNA combination according to claim 1, characterized in that: The steps include: The pYSY-sgRNA plasmid was amplified by PCR using sgRNA2-F, sgRNA5-F, sgRNA6-F, sgRNA8-F, sgRNA13-F and sgRNA16-F in the primer combination of claim 2 and the downstream primer R-Common, and the amplified products were respectively subjected to in vitro transcription to obtain sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16.

4. Use of the sgRNA combination according to claim 1, the primer combination according to claim 2, or the sgRNA combination prepared by the preparation method according to claim 3 in knocking out the zebrafish srsf5b gene and / or constructing a zebrafish srsf5b gene mutant model.

5. A kit for knocking out the zebrafish srsf5b gene and / or constructing a zebrafish srsf5b gene mutant model, characterized in that: The invention comprises the sgRNA combination according to claim 1, the primer combination according to claim 2, or the sgRNA combination prepared by the preparation method according to claim 3.

6. The kit according to claim 5, characterized in that The kit also includes a Cas9 protein.

7. A method for constructing a zebrafish srsf5b gene mutant model, characterized in that: The steps include: Introducing a mixture of an sgRNA combination and a Cas9 protein into zebrafish fertilized eggs, selecting embryos with effective knockout, and culturing them to adult fish to obtain F0 generation mutant zebrafish; the sgRNA combination is the sgRNA combination of claim 1, or the sgRNA combination synthesized using the primer combination of claim 2, or the sgRNA combination prepared by the preparation method of claim 3; The F0 generation mutant zebrafish is hybridized with wild-type zebrafish to obtain F1 generation embryos, the F1 generation embryos are screened for mutant embryos, and the embryos are cultured to adult fish to obtain a zebrafish srsf5b gene mutant model.

8. The construction method according to claim 7, characterized in that: In the mixture of sgRNA combination and Cas9 protein, the concentration of sgRNA combination is 200 ng / μL, and the concentration of Cas9 protein is 400 ng / μL.

9. The construction method according to claim 8, characterized in that: The mass ratio of sgRNA2, sgRNA5, sgRNA6, sgRNA8, sgRNA13 and sgRNA16 in the sgRNA combination is 1:1:1:1:1:

1.

10. The construction method according to claim 7, characterized in that: The method for selecting effective knockout embryos includes the following steps: Fertilized eggs developed to 24 hpf were taken and genomic DNA was prepared; Using the genomic DNA as a template, PCR amplification is performed using upstream amplification primers and downstream amplification primers to obtain a PCR amplification product; The PCR amplification product is compared with the wild-type zebrafish srsf5b gene. If the comparison results are different, the fertilized egg that develops to 24 hpf is an effective knockout embryo; The upstream amplification primer includes the nucleotide sequence shown in SEQ ID NO: 17; The downstream amplification primer includes the nucleotide sequence shown in SEQ ID NO: 18.

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