A method for knocking out phox gene 3 of hypophthalmichthys molitrix and application thereof in creating new germplasm of hypoxia-resistant hypophthalmichthys molitrix

Abstract

The application belongs to the technical field of fish breeding, and discloses a method for knocking out PHD3 gene of Hypophthalmichthys molitrix and application thereof in creating new germplasm of low-oxygen-tolerant Hypophthalmichthys molitrix. Taking PHD3 gene as a target gene, two gRNAs targeting the first exon of PHD3 gene are synthesized by in vitro transcription, mixed with Cas9 protein, and used to edit genes of wild-type Hypophthalmichthys molitrix, so as to improve the knocking-out efficiency of PHD3 gene, and then F0 generation mutants of Hypophthalmichthys molitrix with PHD3 gene knocked out are efficiently obtained. The F0 generation mutants of Hypophthalmichthys molitrix with PHD3 gene knocked out are subjected to low-oxygen stress experiment, and the result shows that the low-oxygen tolerance of the PHD3 knockout mutants of Hypophthalmichthys molitrix is significantly improved compared with the wild type. The application successfully realizes efficient knocking out of PHD3 gene of Hypophthalmichthys molitrix and obtains Hypophthalmichthys molitrix PHD3 mutants with significantly enhanced low-oxygen tolerance, thereby providing an effective technical means for carrying out functional research and genetic improvement of Hypophthalmichthys molitrix, and having important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of fish breeding technology, specifically relating to a method and application of creating hypoxia-tolerant silver carp by efficiently knocking out the PHD3 gene using dual gRNAs. Background Technology

[0002] Silver carp ( Hypophthalmichthys molitrix Widely distributed in various waters of my country, silver carp are a popular economic fish due to their tender flesh and rich nutrition. In recent years, silver carp have rapidly become one of the dominant species in my country's aquaculture due to their advantages such as short food chain, strong disease resistance, and low price. According to the 2025 China Fisheries Statistical Yearbook, my country's silver carp production reached 3.9111 million tons in 2024, ranking second among major freshwater aquaculture fish in my country, making a significant contribution to ensuring a stable and effective supply of aquatic products. Furthermore, silver carp feed on plankton in the water, effectively controlling the proliferation of cyanobacterial blooms. Therefore, in addition to meeting the demand for high-quality protein in my country, silver carp also have a positive impact on improving water quality and purifying water bodies, playing an important role in improving the lake environment.

[0003] Gene editing technology refers to a gene manipulation technique that can perform targeted modifications to the genome of an organism, such as point mutation, gene knockout, simultaneous editing at multiple sites, deletion of small or even large DNA fragments, DNA rearrangement, gene expression activation and deactivation, gene silencing and activation, guided editing, and targeted knock-in of exogenous genes. Since its introduction in 2013, CRISPR / Cas9 technology has been rapidly and widely applied in various fields, including biological breeding. Gene editing technology has been widely used in the study of gene function and trait improvement in economically important fish species in aquaculture, achieving a series of important breakthroughs. However, research progress in gene function, gene editing, and trait improvement in silver carp remains very slow. This is mainly due to the insufficient analysis of the genetic basis of superior traits in silver carp and the limited number of key target genes identified. Furthermore, silver carp have a long sexual maturity cycle, generally requiring more than three years; simultaneously, they are intolerant of low oxygen levels and highly stressed. During artificial breeding, procedures such as artificial spawning induction, egg extraction, and sperm collection can easily lead to the death of silver carp. Meanwhile, the damage caused by microinjection easily leads to the death of silver carp fertilized eggs, resulting in a low hatching rate. In addition, knocking out the target site is prone to off-target effects, resulting in low targeting efficiency. All of these factors severely limit the research on silver carp gene function and the improvement of genetic breeding.

[0004] Using zebrafish, a model organism among cyprinid fish, as the research subject, it was found that knocking out PHD3 can enhance the zebrafish's tolerance to hypoxia. Silver carp is a cyprinid fish with relatively poor tolerance to hypoxia; using gene editing technology to knock out PHD3 in silver carp holds promise for creating new hypoxia-tolerant silver carp germplasm. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the knockout efficiency of the PHD3 gene in silver carp by using two gRNAs in combination and its application in creating new hypoxia-tolerant silver carp germplasm.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method and application for knocking out the PHD3 gene in silver carp: This invention selects the silver carp PHD3 gene (serial number OL795415.1, containing 5 exons), designs two target sites on the first exon of the functional domain of this gene, synthesizes gRNA of the two target genes through in vitro transcription, mixes it with Cas9 protein, and introduces the mixture into silver carp fertilized eggs via microinjection. After hatching, the knockout efficiency of the target sites alone and the two targets in combination in the F0 generation of silver carp is detected, thereby efficiently obtaining F0 generations with knockout effect of the silver carp PHD3 gene. This invention efficiently achieves the knockout of the silver carp PHD3 gene, providing an effective technical means for conducting silver carp breeding and gene function research.

[0008] This invention protects a combination of gRNAs targeting the silver carp PHD3 gene, comprising two gRNAs, the nucleotide sequences of which are shown in SEQ ID NO.1 and 2, and the corresponding target sequences are shown in SEQ ID NO.5 and 6, respectively.

[0009] Another aspect of the present invention protects a knockout kit for the silver carp PHD3 gene, comprising a combination of Cas9 protein and gRNA, the gRNA sequence of which is shown in SEQ ID NO.1 and 2.

[0010] Another aspect of this invention protects a method for knocking out the PHD3 gene in silver carp, comprising injecting a combination of Cas9 protein and gRNA into fertilized eggs at the single-cell stage of silver carp, wherein the gRNA sequences are shown in SEQ ID NO. 1 and 2, and screening for the F0 generation of PHD3 gene knockout by genotyping. Preferably, the injection concentrations of the two gRNAs are 1:1, and the final concentration of each gRNA is 250 ng / μL; the primers used for amplifying the target sequence in genotyping are shown in SEQ ID NO. 3 and 4.

[0011] This invention also protects the application of the above-mentioned gRNA combination in the cultivation of new hypoxia-tolerant silver carp germplasm.

[0012] Two gRNA targets were designed for the first exon of the silver carp PHD3 gene. Target 1 (sequence shown in SEQ ID NO. 5) had an F0 generation knockout efficiency of 60%, and target 2 (sequence shown in SEQ ID NO. 6) had an F0 generation knockout efficiency of 20%. When the two targets were used together, the F0 generation knockout efficiency was 100%, thus significantly improving the knockout efficiency of the silver carp PHD3 gene.

[0013] The F0 generation mutant of silver carp with PHD3 gene knockout constructed by the above method exhibits hypoxia tolerance: In a specific embodiment of the present invention, hypoxia stress experiments were conducted on the F0 generation mutant of silver carp with PHD3 gene knockout. Through phenotypic observation and mortality time recording, it was found that the silver carp with PHD3 knockout mutant has a significantly enhanced hypoxia tolerance compared to wild-type silver carp. Therefore, the silver carp with PHD3 knockout mutant is a potential new hypoxia-tolerant silver carp germplasm.

[0014] Compared with existing technologies, this invention is the first to perform gene editing on the PHD3 gene of silver carp and efficiently obtain F0 generation with knockout effect. The method described in this invention can efficiently obtain silver carp gene knockout strains, providing a method for the genetic improvement of aquatic organisms and having significant scientific value for breeding new varieties with excellent traits such as tolerance to low oxygen levels. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the target sites for knocking out the PHD3 gene in silver carp. Target sites 1 and 2 are located on the first exon of the PHD3 gene, respectively.

[0016] Figure 2 Sequencing diagram of PCR products from the F0 generation of silver carp PHD3 gene editing. In the diagram, triangles indicate the initiation position of the silver carp PHD3 gene mutation.

[0017] Figure 3 This study assesses the hypoxia tolerance of the PHD3 gene-edited F0 generation of silver carp. Compared to wild-type silver carp, the PHD3 gene-edited F0 generation exhibits significantly improved hypoxia tolerance. Red arrows indicate deceased individuals, and green arrows indicate individuals surfacing due to hypoxia. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0019] Example 1: Efficiently obtaining the F0 generation mutant of silver carp PHD3 gene editing

[0020] 1. Design of gRNA targets

[0021] The silver carp PHD3 gRNA target was designed based on the website (https: / / crispor.gi.ucsc.edu / ). The silver carp PHD3 gene sequence in this example was downloaded from the NCBI database, sequence number OL795415.1, and this gene contains 5 exons. The gRNA target design principles were: gRNA targets should be designed in early exons or functional regions to ensure that a fully functional PHD3 protein cannot be synthesized; gRNA targets with high fractions should be selected to ensure they are effective targets; and the gRNA sequence should be linked to PAM (NGG). Based on these design principles, we designed two gRNA targets on the first exon of the silver carp PHD3 gene (e.g., ...). Figure 1 As shown), the target sequence is:

[0022] Target 1: 5'-ggaccgaggctacttttacg-3' (SEQ ID NO.5);

[0023] Target 2: 5'-ggcgaagcagtggagtttgc-3' (SEQ ID NO.6).

[0024] 2. gRNA Synthesis

[0025] The pUC19-gRNA vector (https: / / www.addgene.org / 137776 / ) is used to amplify sgRNA. The primers are as follows:

[0026] Target 1-F: 5'-TAATACGACTCACTATAggaccgaggctacttttacgGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO.7);

[0027] Target 2-F: 5'-TAATACGACTCACTATAggcgaagcagtggagtttgcGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO. 8);

[0028] R: 5'-AAAAGCACCGACTCGGTGCC-3' (SEQ ID NO.9);

[0029] The PCR system consisted of: 20 μL of I5 DNA polymerase (purchased from MCLAB, catalog number I5HM-OEM), 1.6 μL of F primer (concentration of 10 μM), 1.6 μL of R primer (concentration of 10 μM), 15.8 μL of ddH2O, and 1 μL of gRNA plasmid template (concentration of 300 ng / μL).

[0030] The PCR reaction conditions were as follows: 98℃ pre-denaturation for 5 minutes; 98℃ denaturation for 10 seconds, 60℃ annealing for 12 seconds, 72℃ extension for 15 seconds, 35 cycles; and 75℃ final extension for 5 minutes. The PCR products were subjected to 1% agarose gel electrophoresis. After verifying the correct band size, the PCR products were purified and recovered, and the concentration of the recovered products was determined.

[0031] The synthesized gRNA sequence is as follows:

[0032] gRNA1: 5'-TAATACGACTCACTATAggaccgaggctacttttacgGTTTTAGAGCTAGAAATAGCaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttt-3' (SEQ ID NO. 1);

[0033] gRNA2: 5'-TAATACGACTCACTATAggcgaagcagtggagtttgcGTTTTAGAGCTAGAAATAGCaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttt-3' (SEQ ID NO. 2).

[0034] 3. Purification of gRNA

[0035] According to the instructions of the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific, USA), gRNA was transcribed in vitro in a total volume of 20 μL and purified using lithium chloride precipitation. The specific steps for gRNA purification were as follows: 1 μL of DNase I was added to the gRNA in vitro transcription system, and the mixture was incubated in a 37°C water bath for 15 minutes to remove residual DNA. 1 μL of 0.5 M EDTA was added, and the reaction was stopped by standing in a 65°C water bath for 10 minutes. Then, lithium chloride was added to bring the final concentration of lithium chloride in the solution to 2.5 M. The mixture was mixed, and the solution was precipitated at -20°C or -80°C for 2 hours or overnight. The precipitate was collected by centrifugation at 16000×g for 20 minutes at 4°C. 50 μL of pre-chilled 70% ethanol was added to wash the precipitate to remove residual salts. The ethanol was removed by centrifugation at 13000×g for 5 minutes at 4°C. The precipitate was allowed to dry naturally, and 20 μL of DEPC water was added to dissolve the precipitate. Take 1 μL to detect RNA concentration, and then detect gRNA quality by 1% agarose gel electrophoresis. Store at -80℃ for later use.

[0036] 4. Microinjection

[0037] Embryos used for silver carp microinjection were obtained through artificial breeding and fertilization of sexually mature male and female silver carp parents. An injection system was prepared using a mixture of gRNA and Cas9 protein, with a final concentration of 250 ng / μL for each gRNA and 400 ng / μL for Cas9. The mixture was injected into fertilized silver carp eggs spread in a culture dish using a Picoliter Microinjector (Warner, PL-100A, USA). Injection was ideally completed within 2 hours of fertilization. After injection, the fertilized eggs were placed in an incubation tank at a water temperature of 23-28 ℃ for incubation. During incubation, water quality was closely monitored to ensure adequate oxygenation, and dead eggs were removed promptly.

[0038] 5. Detection of knockout efficiency

[0039] Once the F0 generation of gene-edited silver carp reached a size suitable for tail fin identification, genomic DNA was rapidly extracted using an alkaline lysis method. The tail fins were obtained using sterilized blades and tweezers and placed in PCR tubes. 18 μL of 50 mM NaOH was added to each PCR tube, followed by digestion at 95°C for 40 minutes. After cooling, 2 μL of pH 8.0 Tris HCl was added to each PCR tube to neutralize the pH, and the tubes were then stored at 4°C. This completed the preparation of the silver carp tail fin DNA template.

[0040] Subsequently, PCR was performed to amplify the corresponding sequence fragment. The amplified gene fragment was 337 bp in length, and the primer sequences for identification are as follows:

[0041] F: 5'-atgcaccaagctcaagaaaaaatgcc-3' (SEQ ID NO. 3);

[0042] R: 5'-acagagaaatgagtttgtctatgagtgttaataagaaat-3' (SEQ ID NO. 4).

[0043] The PCR system consisted of: 10 μL of 2×SanTaq Fast PCR Master Mix (purchased from Sangon Biotech, catalog number BN32062), 1 μL of F primer (concentration of 10 μM), 1 μL of R primer (concentration of 10 μM), 7 μL of ddH2O, and 1 μL of DNA template.

[0044] The PCR reaction conditions were: 94 ℃ pre-denaturation for 3 minutes; 94 ℃ denaturation for 15 seconds, 60 ℃ annealing for 20 seconds, 72 ℃ extension for 15 seconds, 35 cycles; and a final extension at 72 ℃ for 5 minutes. The PCR products were sequenced and analyzed to obtain peak diagrams. The positions of the double peaks were used to determine the corresponding effective knockout targets, thus identifying the F0 generation with mutation effects in the silver carp PHD3 gene. Statistical analysis showed that the knockout efficiency of target 1 was 60%, the knockout efficiency of target 2 was 20%, and the knockout efficiency of the two targets combined was 100%. Representative results are shown below. Figure 2 As shown in the figure. Knockout efficiency = (Number of tails with bimodal peaks at the target site / Total number of tails edited using the corresponding target site) × 100%.

[0045] Example 2: Silver carp PHD3 gene-edited mutants show significantly enhanced hypoxia tolerance.

[0046] Wild-type silver carp with a body length of 2 cm (±0.5 cm) and PHD3 knockout mutant silver carp were placed in a hypoxia workstation (Ruskinn INVIVO2 I-400) for hypoxia treatment. The oxygen concentration was set to 5%, and the experimental fish were kept in a constant temperature environment of 28℃. The condition of the silver carp was observed every hour, the number of deaths was counted, and photographs were taken for recording. Figure 3 As shown, red arrows indicate dead individuals, and green arrows indicate individuals that have surfaced.

[0047] Experimental results are as follows Figure 3 As shown, after 2 hours of hypoxia treatment, all wild-type silver carp died, while the PHD3 gene-edited mutant silver carp survived. Furthermore, the PHD3 knockout mutant silver carp only showed signs of hypoxia (surfacing for air) after 3 hours of hypoxia treatment and died after 4 hours. This indicates that the PHD3 gene-edited mutant silver carp has significantly enhanced hypoxia tolerance.

Claims

1. A gRNA targeting the PHD3 gene of Hypophthalmichthys nobilis, characterized in that, It includes two gRNAs, the nucleotide sequences of which are shown in SEQ ID NO.1 and 2.

2. A vector comprising the gRNA of claim 1.

3. A kit for knocking out the PHD3 gene in silver carp, characterized in that, It includes a combination of Cas9 protein and gRNA, the gRNA sequence of which is shown in SEQ ID NO.1 and 2.

4. A method for knocking out the PHD3 gene in silver carp, characterized in that, This includes injecting a combination of Cas9 protein and gRNA into fertilized eggs in the single-cell stage of silver carp, wherein the gRNA sequence is shown in SEQ ID NO.1 and 2, and screening for the F0 generation with PHD3 gene knockout by genotyping.

5. The knockout method according to claim 4, characterized in that, The injection concentrations of the two gRNAs were 1:

1.

6. The knockout method according to claim 4, characterized in that, The primers used to amplify the target sequence in the genotype detection are shown in SEQ ID NO.3 and 4.

7. The application of the gRNA described in claim 1 in the cultivation of new hypoxia-tolerant silver carp germplasm.

8. The application of the knockout method according to any one of claims 4 to 6 in the creation of new hypoxia-tolerant silver carp germplasm.

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