Method for knocking out grass carp SMYD3 gene and application

Abstract

The invention relates to the technical field of grass carp (Ctenopharyngodon idella), in particular to a method for knocking out a grass carp SMYD3 gene and application of the grass carp SMYD3 gene. The nucleotide sequence of the gRNA is as shown in SEQ ID NO: 1. The method comprises the following steps: synthesizing gRNA of a target gene through in-vitro transcription, then mixing the gRNA with Cas9 protein, introducing the mixture into an animal pole in a grass carp fertilized egg single-cell period by adopting a microinjection mode, and hatching to obtain the grass carp F0 generation with the target gene mutation. According to the application, the knockout of the grass carp gene is efficiently realized, and an effective technical means is provided for developing grass carp breeding and gene function research.

Description

Technical Field

[0001] The present application relates to the technical field of grass carp (Ctenopharyngodon idellus), and in particular to a method and application of knocking out the SMYD3 gene of grass carp. Background Art

[0002] Grass carp (Ctenopharyngodon idellus) is a herbivorous fish of the Cyprinidae family. Its sexual maturity cycle is generally 5-6 years. The conventional mixed breeding method is used, and the breeding cycle is long and the efficiency is low. At present, there is only one new variety of grass carp approved by the Aquatic Original Breeding Committee, "Husu No. 1" (Announcement of the Ministry of Agriculture and Rural Affairs of the People's Republic of China, No. 836, 2024), which is far from meeting the development needs of the grass carp industry. With the development of high-density intensive breeding, grass carp is often in a low-oxygen environment during the breeding process, resulting in slow growth and decreased immunity, which ultimately causes serious economic losses, so it is urgent to cultivate new varieties suitable for industry needs. Methyltransferase SMYD3 (SET and MYND domain containing3) is a member of the SMYD lysine methylase family. It contains two domains, SET (Su(var)3-9, Enhancer of zeste, Trithorax) and MYND (Myeloid-Nervy-DEAF-1). It was discovered as a member of the RNA polymerase II complex and was first thought to be active on histone H3 lysine amino acid residue 4 (H3K4) (Hamamoto et al., 2004). Later studies found that reducing SMYD3 expression can improve zebrafish's tolerance to hypoxia (Wang et al., 2022).

[0003] Gene editing technology is a technology that can precisely transform the genome of an organism by inserting, knocking out, modifying or replacing specific DNA fragments, achieving site-specific gene knockout and site-specific integration of exogenous genes, and editing the target gene in an organism (Wei Jingliang, Wu Tianwen, Ruan Jinxue, et al. Research progress in improving livestock using genome editing technology [J]. China Agricultural Science and Technology Herald, 2014 (1): 7. DOI: 10.13304 / j.nykjdb.2014.003.). Three genome editing technologies, namely zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspersed short palindromic repeats-associated protein 9 (CRISPR-Cas9), can cut the DNA double helix at the target site, induce the cell's endogenous repair mechanism, and cause homologous recombination repair or non-homologous end joining, thereby improving the efficiency of gene knockout, site-specific integration and replacement (Wang Wei, Hu Hongxia. Research on gene editing technology in fish and prospects for its application in aquaculture [J]. China Fisheries, 2019(09):87-88).

[0004] At present, gene editing technology has been widely used in the improvement of economic fish traits and gene function research (Sun Y, Zheng GD, Nissa M, et al. Disruption of mstna and mstnb gene through CRISPR / Cas9 leads to elevated muscle mass in blunt snoutbream (Megalobrama amblycephala) [J]. Aquaculture, 2020, 528: 735597. DOI: 10.1016 / j.aquaculture.2020.735597). At present, only Gao Zexia et al. (2022) successfully obtained the grass carp run2b gene mutant strain using the CRISPR-Cas9 method (invention patent: CN116515907A). The research progress of grass carp gene function and genetic improvement breeding has been slow, which has been constrained by the lack of effective gene editing methods, which has seriously restricted the breeding and gene function research process. Summary of the invention

[0005] The present application provides a method and application of knocking out the SMYD3 gene in grass carp, which is applied to the functions of genes related to the efficient use of dissolved oxygen in grass carp and genetic improvement breeding. Among them, the grass carp SMYD3 gene (GeneID: 127498386, the gene is 182730bp in length, including 12 exons and 11 introns), the target site gRNA sequence is designed in the sixth exon of the gene, and then mixed with the Cas9 protein, and the mixture is introduced into the animal pole of the single-cell stage of the grass carp fertilized egg by microinjection. After hatching, the grass carp F0 generation with target gene mutation can be obtained, and the homozygotes with SMYD3 deletion are obtained by breeding and cultivating for multiple generations. The present application efficiently realizes the knockout of the SMYD3 gene of grass carp, and provides an effective technical means for carrying out breeding and gene function research of grass carp.

[0006] In a first aspect, the embodiment discloses a target site gRNA nucleotide sequence targeting the grass carp (Ctenopharyngodon idellus) SMYD3 gene, and its nucleotide sequence is shown in SEQ ID NO:1.

[0007] In an embodiment of the first aspect, the gRNA targets the sixth exon of the SMYD3 gene.

[0008] In a second aspect, the embodiments disclose a grass carp (Ctenopharyngodon idellus) SMYD3 gene knockout composition, which includes the gRNA and Cas9 protein described in the first aspect.

[0009] In a third aspect, the embodiments disclose a grass carp (Ctenopharyngodon idellus) SMYD3 gene knockout kit, which includes the gRNA and Cas9 protein described in the first aspect.

[0010] In a fourth aspect, the embodiment discloses a method for knocking out the SMYD3 gene of grass carp (Ctenopharyngodon idellus), which comprises injecting the gRNA as described in SEQ ID NO: 1 and the Cas9 protein into the fertilized egg of the grass carp in the single-cell stage.

[0011] In an embodiment of the fourth aspect, the injection concentration of the gRNA is 2000 ng / μL, and the final injection concentration of the Cas9 mRNA is 500 ng / μL.

[0012] In an embodiment of the fourth aspect, mutants are screened from offspring of the grass carp.

[0013] In the fifth aspect, the embodiment discloses a method for preparing the gRNA described in the first aspect. The preparation method comprises: designing and synthesizing a primer pair for the gRNA; performing PCR amplification on the primer pair; and recovering the gRNA from the PCR amplification product.

[0014] In an embodiment of the fifth aspect, the primer pair used to obtain the gRNA as described in SEQ ID NO: 1 includes an upstream primer as described in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO: 3.

[0015] In the sixth aspect, the embodiments disclose the application of the gRNA described in the first aspect in breeding grass carp mutant strains.

[0016] The gRNA, gene knockout composition, kit, method and application provided in the embodiment designed a gRNA target site for the sixth exon of the grass carp SMYD3 gene, and the knockout efficiency in the P0 generation reached 86.67%, which is a high knockout efficiency.

[0017] The screening method provided in the embodiment can be used to obtain a genetically mutant strain of grass carp. The method is simple and easy to operate. Grass carp (Ctenopharyngodon idellus) generally matures sexually for 5-6 years. The creation of new germplasm of hypoxia-resistant grass carp using traditional breeding methods such as group selection and family selection has a long cycle and low efficiency. The gRNA, composition, kit and knockout method provided in the embodiment can knock out the target gene SMYD3 during the fertilized egg period of grass carp, and obtain a group with altered hypoxia-resistant traits by destroying gene expression, thereby achieving precise editing of specific gene sequences, and obtaining new germplasm in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the sequence of the target site of the grass carp SMYD3 gene.

[0019] Figure 2 The following is a sequence diagram of the PCR product of the SMYD3 gene at the mutation site of the mutant grass carp cultivated into the parent F0 generation. In the figure, the square represents the target sequence of the SMYD3 gene, and the triangle represents the position of the SMYD3 mutation. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0021] 1. Experimental Materials

[0022] Wild grass carp male and female parents were raised at the Yaowan Experimental Plant of Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. Embryos used for microinjection of grass carp were obtained from semi-artificial breeding spawning of sexually mature male and female parents.

[0023] 2. Design of guide RNA (gRNA)

[0024] The design of gRNA target site needs to download the DNA sequence of grass carp SMYD3 gene (127498386) from NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / 127498386), and then screen the appropriate target site of the target gene on the website (https: / / www.crisprscan.org); the gRNA target site of the target gene is preferably designed in the exon or functional region; select the target site with a higher score to ensure that it is an effective target; ensure that the target sequence is connected to PAM (NGG); in addition, the target of the designed target gene can be tested for GC content, Tm value and secondary structure through the website (http: / / www.oligoevaluator.com / LoginServlet). The sequence of the synthetic gRNA target is composed of 17bp T7 promoter, 20bp target sequence and 15bp scaffoldoverlap sequence: (TAATACGACTCACTATAggNNNNNNNNNNNNNNNNNNGTTTTAGAGCTAGAA).

[0025] The target site gRNA sequence in this embodiment is located on the sixth exon of the grass carp SMYD3 gene; the target site gRNA sequence is: 5'-TGGAGAGCAGCAGTACGTGG-3', SEQ ID NO: 1.

[0026] 4. Primers for synthesizing gRNA

[0027] In some embodiments, gRNA can be obtained by designing and synthesizing primers and performing PCR amplification with these primers.

[0028] Upstream primer containing target site gRNA specific to T7 promoter: 5'-TAATACGACTCACTATATGGAGAGCAGCAGTACGTGGGTTTTAGAGCTAGAAATAGC-3', SEQ ID NO: 2

[0029] Downstream primer of gRNA:

[0030] 5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3', SEQ ID NO: 3

[0031] 5. Synthesize gRNA

[0032] The above primers were used for PCR amplification. The PCR system was as follows: 2.5 μL of the upstream primer of 10 μM gRNA, 2.5 μL of the downstream primer of 10 μM gRNA, and 25 μL PCR Master Mix, 20 μL double distilled water, amplify 2 tubes of gRNA totaling 100 μL. The PCR amplification program is 95℃ pre-denaturation for 30 min, 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ extension for 5 min and then stored at 4℃. PCR products were detected by 1.5% agarose gel electrophoresis.

[0033] 6. PCR product recovery and purification

[0034] After detection, the PCR product was purified and recovered using the Omega agarose gel recovery kit, and the concentration was measured using Nanodrop 2000 (ThermoScientific, USA). According to the instructions of the Transcriptaid T7 high yield transcription kit (Thermo Scientific, USA), RNA was transcribed in vitro and purified and recovered by lithium chloride precipitation to obtain gRNA. 1 μL was taken out to measure the RNA concentration, and the quality of RNA was tested by 1.5% agarose gel electrophoresis and then stored in a -80°C refrigerator for use.

[0035] 7. Microinjection

[0036] Before injection, male and female grass carp parents were placed in a breeding parent pool at a ratio of 2:1, and female parents were injected with luteinizing hormone-releasing hormone A2 (0.4 μg / kg) for induced labor, and the injection amount of male parents was half of that of female parents. The effect time after injection of the oxytocin is generally 10 minutes (after the embryo absorbs water and the fertilized egg develops to the single-cell stage, the injection begins). The injection system was prepared by mixing gRNA and Cas9 protein (GMP-CA9S18, AcroBIOSYSTEMS), in which the final concentration of gRNA was 2000 ng / μL, the concentration of Cas9 protein was 500 ng / μL, and phenol red was added with a final concentration of 0.2% as an indicator. The experimental reagents were injected into the grass carp embryos at the single-cell stage using a Picoliter Microinjector (Warner, PL-100A, USA). After the injection, the grass carp embryos were cultured in still pond water at a temperature of 25±2°C. During the incubation process of grass carp embryos, special attention should be paid to water quality management. The water should be kept with sufficient oxygen, and the water should be changed in time and eggs that have stopped developing should be cleaned up to prevent hypoxia or the occurrence of saprolegniasis.

[0037] 8. Detection of target mutation rate

[0038] Select the surviving young fish, cut their tail fins, and use the alkaline lysis method to quickly extract the grass carp genomic DNA. Place the fins in a 0.2ml eight-tube strip, add 100μL NaOH, and perform PCR reaction at 94℃ for 20min, then end the reaction at 4℃ for 20min. Vortex and mix well, add 5μL Tris-Hcl, mix well, and store at 4℃. This can be used as a DNA template. The amplification primers are:

[0039] SMYD3-F:5'-CAGTGATGCTTTTTGTAGCAAGC-3', SEQ ID NO:4

[0040] SMYD3-R:5'-GTCTGGTATCACCAAATCATGAG-3', SEQ ID NO:5.

[0041] The amplification PCR system is: 1.5 μL each of upstream and downstream primers, PCR Master Mix 25μL, DNA template 2μL, enzyme-free water to 50μL. PCR amplification program is 94℃ pre-denaturation for 3min; 94℃ denaturation for 30s, 58℃ annealing for 30s, 72℃ extension for 30s, 32 cycles; 72℃ extension for 5min and then stored at 4℃. PCR products were sent to Qingke Bio for sequencing analysis. Figure 2The sequencing results shown in the figure show that there are double peaks at the mutation site, and the silver carp with mutations are screened out and cultivated into the parent F0 generation. The knockout efficiency of the F0 generation reaches 86.67%, which is a high knockout efficiency. Among them, the knockout efficiency = the tail number of double peaks at the target site / the total number × 100%.

[0042] 9. Obtaining a stable grass carp gene mutation strain

[0043] The mutated grass carp is cultivated into the parental F0 generation; continuing to breed and cultivate for many generations to obtain homozygous SMYD3 deficiency is a stable mutant strain.

[0044] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A gRNA targeting the SMYD3 gene of grass carp (Ctenopharyngodon idellus), the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. The gRNA according to claim 1, which targets the sixth exon of the SMYD3 gene.

3. A knockout composition for the grass carp (Ctenopharyngodon idellus) SMYD3 gene, comprising the gRNA and Cas9 protein as described in claim 1 or 2.

4. A knockout kit for the grass carp (Ctenopharyngodon idellus) SMYD3 gene, comprising the gRNA and Cas9 protein as claimed in claim 1 or 2.

5. A method for knocking out the SMYD3 gene of grass carp (Ctenopharyngodon idellus), comprising injecting the gRNA described in SEQ ID NO: 1 and the Cas9 protein into a fertilized egg of the grass carp in the single-cell stage.

6. The knockout method according to claim 5, wherein the injection concentration of the gRNA is 2000 ng / μL, and the final injection concentration of the Cas9 protein is 500 ng / μL.

7. The knockout method according to claim 5 or 6, wherein mutants are screened from the offspring of the grass carp, wherein: The PCR amplification primer pair used for screening mutants includes an upstream primer as shown in SEQ ID NO:4 and a downstream primer as shown in SEQ ID NO:

5.

8. The method for preparing gRNA according to claim 1 or 2, comprising: Design and synthesize primer pairs for gRNA; Performing PCR amplification on the primer pair; The gRNA is recovered from the PCR amplified product.

9. The preparation method according to claim 8, wherein: The primer pair used to obtain the gRNA as described in SEQ ID NO: 1 includes an upstream primer as described in SEQ ID NO: 2 and a downstream primer as shown in SEQ ID NO:

3.

10. Use of the gRNA according to claim 1 or 2 in cultivating a mutant strain of grass carp.

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