A method for breeding a crucian carp strain without intermuscular spinules

Abstract

A method for breeding a crucian carp strain with no intermuscular spines is disclosed, relating to a method for breeding aquaculture varieties. To genetically improve the intermuscular spines of crucian carp, the present invention proposes a method for breeding crucian carp with fewer or no intermuscular spines by genetically knocking out the bmp6 gene. The breeding method involves designing knockout target sites for each of the two copies of the bmp6 gene, bmp6a and bmp6b, in the crucian carp genome. Then, through two rounds of gene knockout and screening, an F2 generation homozygous line with double mutations of bmp6a and bmp6b is obtained. This F2 generation homozygous line with double mutations of bmp6a and bmp6b is then used for propagation to create a new crucian carp strain with no intermuscular spines. The method of the present invention yields a crucian carp strain with fewer than 20 intermuscular spines and no intermuscular spines.

Description

Technical Field

[0001] The invention relates to a method for cultivating aquaculture varieties. Background Art

[0002] Cyprinidae are the primary farmed fish species in my country, with an annual production of over 20 million tons, providing nearly one-third of the nation's high-quality animal protein. However, the high number of intermuscular spines in cyprinidae species can cause inconvenience during consumption and can even lead to physical harm, such as choking. This also hinders the processing of fish products (such as fish balls). Therefore, genetic improvement of intermuscular spines in cyprinidae species has become a key goal of aquatic genetics and breeding in my country. There are several reports on intermuscular spine selection. For example, Xu Xiaofeng et al. (2015) discovered a normal-growing mutant with no intermuscular spines in a gynogenetic population of grass carp, but no reports have been obtained of mutant populations with no or few intermuscular spines. Brazilian scientists (2017) used X-rays to screen for a population lacking intermuscular spines in the giant carp (Pacu maximus), but subsequent breeding efforts have also failed to yield populations with few or no intermuscular spines (Stokstad, 2020). Guo et al. (2018) conducted a hybridization experiment between Megalobrama terminalis and Culter alburnus and found that the number of intermuscular spines (127) in the offspring of the hybrid of Megalobrama terminalis and Culter alburnus was less than that of the father (137) and greater than that of the mother (124), but significantly less than that of the offspring of the hybrid of Megalobrama terminalis and Culter alburnus (133). This indicates that hybridization can produce varieties with fewer intermuscular spines, and they applied for an invention patent (publication number CN107347747A). Li Ling et al. (2013) reported that the number of intermuscular spines in artificially bred hybrid crucian carp was less than that in wild crucian carp, and that the number of intermuscular spines in crucian carp could be reduced through artificial breeding and hybridization. Bao Baolong et al. developed a method to thicken intermuscular spines by knocking out the MSTN gene (Patent Publication Nos. CN111560401A, CN111549030A, CN111549031A, and CN111500581A). Gao Zexia et al. generated mutants with a greater than 70% reduction in intermuscular spines by knocking out the scxa gene in zebrafish. However, this also resulted in dysplasia of other bones, such as the ribs (Patent Application Nos. 201911104496.6; Nie et al., 2020; Kague et al., 2019). There are no reports of this method being applied to other cyprinid species. In summary, while there are some methods for reducing the number of intermuscular spines in cyprinids, no new species or strains of cyprinids have yet to be developed.

[0003] Crucian carp is one of my country's major aquaculture species, with an annual production of approximately 3 million tons. Its delicious meat is deeply loved by the public. However, its quality is also affected by the number of intermuscular spines (the average number of which varies from species to species is approximately 71 to 84), making intermuscular spines a key target in genetic breeding. While there have been reports of reducing the number of intermuscular spines through hybrid breeding and artificial selection, no strain or variety has been found that has achieved a reduction of more than 50%. Summary of the Invention

[0004] In order to genetically improve the intermuscular spines of crucian carp, the present invention provides a method for breeding crucian carp with fewer or no intermuscular spines by knocking out the bmp6 gene.

[0005] The method for cultivating a crucian carp strain without intermuscular thorns of the present invention is carried out by cultivating a crucian carp strain without intermuscular thorns according to the following steps:

[0006] Knockout target sites were designed for the two copies of the bmp6 gene in the crucian carp genome, bmp6a and bmp6b. Then, through two rounds of gene knockout and screening, an F2 generation homozygous line with bmp6a and bmp6b double gene mutations was obtained. This F2 generation homozygous line of bmp6a and bmp6b double gene mutations was then used to propagate a new strain of crucian carp without intermuscular spinules.

[0007] Among them, the corresponding sgRNA on bmp6a or bmp6b or bmp6a and bmp6b is mixed with Cas9 protein and then microinjected into one-cell stage embryos of crucian carp to perform the first round of gene knockout, and the F0 generation population is constructed. The F0 generation population is then cultured for 3 to 5 months for PIT labeling and DNA extraction, and then sequencing is performed to determine the alleles and mutation rates of somatic cell mutations in individual crucian carp. F0 generation individuals with somatic cell mutation lines and a somatic cell mutation rate of more than 95% are selected as parents to prepare 0 generation fertilized eggs;

[0008] The corresponding sgRNA on bmp6a or bmp6b or bmp6a and bmp6b is mixed with Cas9 protein and microinjected into 0-generation fertilized eggs for a second round of gene knockout to construct an F1 generation population. The F1 generation population is then cultured for 3 to 5 months for PIT labeling and DNA extraction, and then sequencing is performed to determine the alleles and mutation rates of somatic mutations in individual crucian carp. The F1 generation with a somatic bmp6a and bmp6b double gene mutation line and a somatic mutation rate of more than 95% is selected as the parent for breeding to construct the F2 generation, and then the bmp6a and bmp6b double gene mutation homozygous line is selected from the F2 generation.

[0009] The method of the invention obtains crucian carp varieties with less than 20 intermuscular spines or no intermuscular spines.

[0010] Because the crucian carp genome has undergone the fourth round of genome duplication event (Chen et al. 2019), there are two copies of the bmp6 gene in the crucian carp genome. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a comparison diagram of typical individual bone staining observation in Example 1, where the red arrows point to intermuscular thorns;

[0012] Figure 2 This is an X-ray image of the skeleton of a wild-type crucian carp in Example 1, in which the red arrows indicate intermuscular spines;

[0013] Figure 3 This is an X-ray image of the tail skeleton of the mutant in Example 1. It can be seen from the image that there are no intermuscular spines in the tail muscle tissue;

[0014] Figure 4 This is an X-ray image of the trunk skeleton of the mutant in Example 1. It can be seen from the image that there are no intermuscular spines in the dorsal muscle tissue of the trunk;

[0015] Figure 5 This is a diagram of the sequencing results of exon 1 of the new crucian carp strain bmp6a in Example 1; wherein CAA-1, CAA-2, and CAA-3 are target sites of exon 1 of bmp6a; WT is the wild type, the numbers in the sequence number are the mutant PIT marker numbers, and the letters are the mutant allele numbers;

[0016] Figure 6 This is a diagram of the sequencing results of exon 1 of the new crucian carp strain bmp6b in Example 1; wherein CAA-7, CAA-8, and CAA-9 are target sites of bmp6b exon 1; WT is the wild type, the numbers in the sequence number are the mutant PIT marker numbers, and the letters are the mutant allele numbers;

[0017] Figure 7 1 is a comparison diagram of the protein sequence of exon 1 of the new strain crucian carp bmp6a in Example 1; wherein WT is the wild type, the numbers in the sequence number are the mutant PIT marker numbers, and the letters are the mutant allele numbers;

[0018] Figure 8 This is a comparison diagram of the exon 1 protein sequence of the new strain crucian carp bmp6b in Example 1; wherein WT is the wild type, the numbers in the sequence number are the mutant PIT marker numbers, and the letters are the mutant allele numbers. DETAILED DESCRIPTION

[0019] The present invention will be further described in detail below with reference to examples. The following examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0020] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods, and instruments used are conventional in the art and can be obtained commercially by those skilled in the art unless otherwise specified.

[0021] Specific embodiment 1: The method for cultivating crucian carp strain without intermuscular spiny nails in this embodiment:

[0022] Knockout target sites were designed for the two copies of the bmp6 gene in the crucian carp genome, bmp6a and bmp6b. Then, through two rounds of gene knockout and screening, an F2 generation homozygous line with bmp6a and bmp6b double gene mutations was obtained. This F2 generation homozygous line of bmp6a and bmp6b double gene mutations was then used to propagate a new strain of crucian carp without intermuscular spinules.

[0023] Among them, the corresponding sgRNA on (bmp6a) or (bmp6b) or (bmp6a and bmp6b) is mixed with Cas9 protein and microinjected into one-cell stage embryos of crucian carp to perform the first round of gene knockout, and the F0 generation population is constructed. The F0 generation population is then cultured for 3 to 5 months for PIT labeling and DNA extraction, and then sequencing is performed to determine the alleles and mutation rates of somatic mutations in individual crucian carp. F0 generation individuals with somatic mutation lines and a somatic mutation rate of more than 95% are selected as parents to prepare 0 generation fertilized eggs;

[0024] The corresponding sgRNA on bmp6a or bmp6b or bmp6a and bmp6b is mixed with Cas9 protein and microinjected into 0-generation fertilized eggs for a second round of gene knockout to construct an F1 generation population. The F1 generation population is then cultured for 3 to 5 months for PIT labeling and DNA extraction, and then sequencing is performed to determine the alleles and mutation rates of somatic mutations in individual crucian carp. The F1 generation with a somatic bmp6a and bmp6b double gene mutation line and a somatic mutation rate of more than 95% is selected as the parent for breeding to construct the F2 generation, and then the bmp6a and bmp6b double gene mutation homozygous line is selected from the F2 generation.

[0025] Wherein, the DNA extraction and sequencing method is: 2Add 100 μl of lysis buffer (lysate composition: Proteinase K 0.5 mg / ml, Tris (pH 8.0) 10 mM, KCl 50 mM, 0.3% Tween 20, 0.3% NP40) to the fin ray sample. Lysis method: Set the PCR instrument to 55°C for 6 hours and 98°C for 10 minutes. After lysis, mix well, centrifuge at 1000-2000 rpm for 2 minutes, and use the supernatant as a PCR amplification template. PCR amplification: PCR amplification is performed using two pairs of primers: one pair of target site primers (primer sequences for amplifying the target fragment and the 5'-end M13 universal primer sequence shown in Table 2), and the other pair of primers are index primers (M13 universal primer sequence and the 5'-end index base sequence used to identify the sample). A two-step PCR amplification method was used. In the first step, the PCR amplification system consisted of 10 μL of genomic DNA supernatant, 0.5 μL of 1 μM upstream and downstream target primers, and 5 μL of 2× Dream Taq Master Mix (Thermo Fisher, CA, USA), which was supplemented to 10 μL with enzyme-free water. The PCR program was 95°C for 3 min, 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, for 10 cycles, and 72°C for 2 min. In the second step, 0.5 μL of 5 μM upstream and downstream index primers were added to the PCR amplification product of the first step. The PCR program was set to 95°C for 2 min, 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, for 6 cycles, and 95°C for 30 s, 72°C for 30 s, for 15 cycles, and 72°C for 2 min. After PCR amplification, equal amounts of PCR products were mixed and DNA sequencing libraries were constructed using the TrueSeq library construction kit. 300-bp paired-end sequencing was performed on the Illumina MiSeq sequencing platform. High-throughput sequencing data were first used to identify amplification sites and samples using the method described by Tong et al. (2018). The CRISPResso2 program (Fiume et al. 2019) was then used to analyze the read length data for each PCR product in each sample to determine the alleles and mutation rates of somatic mutations.

[0026] Mutant detection was performed using Sanger sequencing. Samples were amplified by PCR using the primers listed in Table 2. The system setup was 25 μL: 2 μL of genomic DNA supernatant, 1 μL each of 10 μM upstream and downstream target primers, 12.5 μL of 2× DreamTaq Master Mix (Thermo Fisher, CA, USA), and enzyme-free water to 25 μL. The PCR reaction program was 95°C for 3 min, 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, for 35 cycles, and finally 72°C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis. After detection, the samples were purified and recovered for TA cloning. After colony PCR amplification, 2 μL of the PCR product was collected and analyzed using 8% polyacrylamide. Colonies with band sizes different from the control were selected for Sanger sequencing.

[0027] Specific embodiment 2: The difference between this embodiment and specific embodiment 1 is that the knockout target sites of the bmp6 gene are shown in Table 1.

[0028] Table 1

[0029]

[0030]

[0031] The rest is the same as the first specific implementation method.

[0032] Table 2

[0033]

[0034] Specific embodiment three: The difference between this embodiment and specific embodiment one or two is that: the sgRNA upstream primer shown in Table 1 and the sgRNA downstream primer with the sequence of 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3' are used to synthesize sgRNA in vitro; the sgRNA in vitro amplification system is: 2.5 μL each of 10 μM sgRNA upstream and downstream primers, 25 μL of 2×Dream Taq Master mix (ThermoFisher, CA, USA), and enzyme-free water to 50 μL; a total of 100 μL is amplified in 2 tubes; the sgRNA in vitro amplification program is denaturation at 95°C for 3 minutes, followed by 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 seconds, and extension at 72°C for 5 minutes; the amplified product is then purified and recovered, and then in vitro transcription is performed, and a 30 μl reaction system is established for each target site: sgRNA 1 μg of PCR product was recovered, 10 μL of NTP Buffer Mix, and 2 μL of T7 RNA Polymerase Mix were added to a volume of 30 μL with enzyme-free water. Transcribe at 37°C for 4 hours. After the reaction, 20 μL of enzyme-free water was added, mixed thoroughly, and 2 μL of DNase I was added. Unreacted DNA was removed by digestion at 37°C for 15 minutes. Other procedures were the same as in Specific Embodiments 1 or 2.

[0035] The sgRNA in vitro PCR product was detected by 1.5% agarose gel electrophoresis, yielding a 120-bp band. After detection, the PCR product was purified and recovered using a PCR product purification and recovery kit (Exygen). The concentration was determined using the Qubit 3 kit (Thermo Fisher, CA, USA) and was subsequently used. The recovered sgRNA PCR product concentration ranged from 100 to 160 ng / μl.

[0036] The in vitro transcribed sgRNA was purified and recovered using an RNA purification kit (Qiagen). The recovered product concentration was determined using the Qubit 3 kit (Thermo Fisher, CA, USA) and stored at -80°C until use. The recovered in vitro transcribed sgRNA concentration ranged from 800 to 3000 ng / μL.

[0037] Specific embodiment 4: This embodiment differs from specific embodiments 1, 2, or 3 in that the microinjection method is as follows: in vitro synthesized sgRNA and Cas9 protein (NEB M0646, MA, USA) are mixed at a molar concentration ratio of 3:1, incubated at room temperature for 10 minutes, and then injected into one-cell crucian carp embryos after adding 25% phenol red. Equal amounts of sgRNA targeting the target site on each exon are mixed and injected, with the final concentration of each sgRNA being no less than 50 ng / μL. The control group is injected with 25% phenol red, and the injection volume per fertilized egg is 1 nL ± 0.02 nL. Other procedures are the same as specific embodiments 1, 2, or 3.

[0038] Example 1

[0039] Method for breeding crucian carp strain without intermuscular spines:

[0040] Knockout target sites were designed for the two copies of the bmp6 gene in the crucian carp genome, bmp6a and bmp6b. Then, through two rounds of gene knockout and screening, an F2 generation homozygous line with bmp6a and bmp6b double gene mutations was obtained. This F2 generation homozygous line of bmp6a and bmp6b double gene mutations was then used to propagate a new strain of crucian carp without intermuscular spinules.

[0041] Among them, the corresponding sgRNA on (bmp6a) or (bmp6b) or (bmp6a and bmp6b) is mixed with Cas9 protein and microinjected into one-cell stage embryos of crucian carp to perform the first round of gene knockout, and the F0 generation population is constructed. The F0 generation population is then cultured for 3 to 5 months for PIT labeling and DNA extraction, and then sequencing is performed to determine the alleles and mutation rates of somatic mutations in individual crucian carp. F0 generation individuals with somatic mutation lines and a somatic mutation rate of more than 95% are selected as parents to prepare 0 generation fertilized eggs;

[0042] The corresponding sgRNA on bmp6a or bmp6b or bmp6a and bmp6b is mixed with Cas9 protein and microinjected into 0-generation fertilized eggs for a second round of gene knockout to construct an F1 generation population. The F1 generation population is then cultured for 3 to 5 months for PIT labeling and DNA extraction, and then sequencing is performed to determine the alleles and mutation rates of somatic mutations in individual crucian carp. The F1 generation with a somatic bmp6a and bmp6b double gene mutation line and a somatic mutation rate of more than 95% is selected as the parent for breeding to construct the F2 generation, and then the bmp6a and bmp6b double gene mutation homozygous line is selected from the F2 generation.

[0043] Wherein, the DNA extraction and sequencing method is: 2Add 100 μl of lysis buffer (lysate composition: 0.5 mg / ml proteinase K, 10 mM Tris (pH 8.0), 50 mM KCl, 0.3% Tween 20, 0.3% NP40) to the fin ray sample. Lysis method: Set the PCR instrument to 55°C for 6 hours and 98°C for 10 minutes. After lysis, mix well, centrifuge at 1000-2000 rpm for 2 minutes, and use the supernatant as a PCR amplification template. PCR amplification: PCR amplification is performed using two pairs of primers: one pair of target site primers (primer sequences for amplifying the target fragment and the 5'-end M13 universal primer sequence shown in Table 2), and the other pair of primers are index primers (M13 universal primer sequence and the 5'-end index base sequence used to identify the sample). A two-step PCR amplification method was used. In the first step, the PCR amplification system consisted of 10 μL of genomic DNA supernatant, 0.5 μL of 1 μM upstream and downstream target primers, and 5 μL of 2× Dream Taq Master Mix (Thermo Fisher, CA, USA), which was supplemented to 10 μL with enzyme-free water. The PCR program was 95°C for 3 min, 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, for 10 cycles, and 72°C for 2 min. In the second step, 0.5 μL of 5 μM upstream and downstream index primers were added to the PCR amplification product of the first step. The PCR program was set to 95°C for 2 min, 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, for 6 cycles, and 95°C for 30 s, 72°C for 30 s, for 15 cycles, and 72°C for 2 min. After PCR amplification, equal amounts of PCR products were mixed and DNA sequencing libraries were constructed using the TrueSeq library construction kit. 300-bp paired-end sequencing was performed on the Illumina MiSeq sequencing platform. High-throughput sequencing data were first used to identify amplification sites and samples using the method described by Tong et al. (2018). The CRISPResso2 program (Fiume et al. 2019) was then used to analyze the read length data for each PCR product in each sample to determine the alleles and mutation rates of somatic mutations.

[0044] Mutant detection was performed using Sanger sequencing. Samples were amplified by PCR using the primers listed in Table 2. The system setup was 25 μL: 2 μL of genomic DNA supernatant, 1 μL each of 10 μM upstream and downstream target primers, 12.5 μL of 2× DreamTaq Master Mix (Thermo Fisher, CA, USA), and enzyme-free water to 25 μL. The PCR reaction program was 95°C for 3 min, 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s, for 35 cycles, and finally 72°C for 5 min. PCR products were detected by 1.5% agarose gel electrophoresis. After detection, the samples were purified and recovered for TA cloning. After colony PCR amplification, 2 μL of the PCR product was collected and analyzed using 8% polyacrylamide. Colonies with band sizes different from the control were selected for Sanger sequencing.

[0045] Among them, the knockout target sites of the bmp6 gene are shown in Table 1.

[0046] The sgRNA upstream primer shown in Table 1 and the sgRNA downstream primer with the sequence of 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3' were used for in vitro synthesis of sgRNA. The sgRNA in vitro amplification system was as follows: 2.5 μL each of 10 μM sgRNA upstream and downstream primers, 25 μL of 2× DreamTaq Master mix (Thermo Fisher, CA, USA), and enzyme-free water to 50 μL. Two tubes were amplified for a total of 100 μL. The sgRNA in vitro amplification program was denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and extension at 72°C for 5 min. The amplified products were then purified and recovered, and then in vitro transcription was performed. A 30 μL reaction system was established for each target site: 1 μg of sgRNA PCR recovered product, NTP Buffer Mix, and 1 μg of PCR product. 10 μL, T7 RNA Polymerase Mix 2 μL, add enzyme-free water to 30 μL; transcribe at 37°C for 4 hours, add 20 μL enzyme-free water at the end of the reaction, mix well, add 2 μL DNase I, and digest at 37°C for 15 minutes to remove unreacted DNA.

[0047] The sgRNA in vitro PCR product was detected by 1.5% agarose gel electrophoresis, yielding a 120-bp band. After detection, the PCR product was purified and recovered using a PCR product purification and recovery kit (Exygen). The concentration was determined using the Qubit 3 kit (Thermo Fisher, CA, USA) and was subsequently used. The recovered sgRNA PCR product concentration ranged from 100 to 160 ng / μl.

[0048] The in vitro transcribed sgRNA was purified and recovered using an RNA purification kit (Qiagen). The recovered product concentration was determined using the Qubit 3 kit (Thermo Fisher, CA, USA) and stored at -80°C until use. The recovered in vitro transcribed sgRNA concentration ranged from 800 to 3000 ng / μL.

[0049] The microinjection method is as follows: in vitro synthesized sgRNA and Cas9 protein (NEB M0646, MA, USA) are mixed at a molar concentration ratio of 3:1, incubated at room temperature for 10 minutes, and then injected into one-cell crucian carp embryos after adding 25% phenol red. Among them, equal amounts of target site sgRNA on each exon are mixed and injected, and the final concentration of each sgRNA is not less than 50 ng / μL. The control group is injected with 25% phenol red, and the injection volume per fertilized egg is 1nL±0.02nL.

[0050] The diploid crucian carp used in this example was obtained from the Hulan Experimental Field of Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences. 2 The fish were reared in a pond and injected with oxytocin (luteinizing hormone-releasing hormone analogue A2 4ug / kg; dioxin maleate 1mg / k, chorionic villus hormone (HCG) 100 units / kg) during breeding. After artificial insemination, the fertilized eggs were incubated in a 24cm×20cm×24cm incubator at a water temperature of 22℃~23℃. After hatching, the fry were fed Artemia spp. 4 times a day after the yolk was absorbed. When the fry reached 2cm~3cm in size in the incubator, they were moved outdoors 500m away. 2 The fish were reared in fish ponds and fed with artificial feed twice a day during the fingerling rearing stage.

[0051] The new strain of crucian carp without intermuscular spines formed in this embodiment was subjected to mutant bone staining observation, and it was found that the intermuscular spines in 5 tail samples were completely missing, and the number of intermuscular spines in 4 tail samples was only 4 to 20 (such as Figures 1 to 4 Detection of the above samples at exon 1 of bmp6a and exon 1 of bmp6b revealed mutations at the three target sites of exon 1 (e.g. Figure 5 and Figure 6 ), gene knockout caused premature termination of protein translation (as shown in Figure 7 and Figure 8 shown).

Claims

1. A method for breeding a crucian carp strain without intramuscular spinules, characterized in that Follow the steps below to breed a crucian carp strain without intermuscular spinules: Knockout target sites were designed for the two copies of the bmp6 gene in the crucian carp genome, bmp6a and bmp6b. Then, through two rounds of gene knockout and screening, an F2 generation homozygous line with bmp6a and bmp6b double gene mutations was obtained. This F2 generation homozygous line of bmp6a and bmp6b double gene mutations was then used to propagate a new strain of crucian carp without intermuscular spinules. The first round of gene knockout was performed by microinjecting a mixture of bmp6a or bmp6b or the corresponding sgRNA on both bmp6a and bmp6b with Cas9 protein into one-cell stage embryos of crucian carp. The F0 generation population was then cultured for 3-5 months for PIT labeling and DNA extraction. Sequencing was then performed to determine the alleles and mutation rates of somatic mutations in individual crucian carp. F0 generation individuals with somatic mutation lines and a somatic mutation rate of more than 95% were selected as parents to prepare 0 generation fertilized eggs. A mixture of sgRNA corresponding to bmp6a or bmp6b, or both bmp6a and bmp6b, and Cas9 protein was microinjected into generation 0 fertilized eggs for a second round of gene knockout, and an F1 generation population was constructed. The F1 generation population was then cultured for 3–5 months for PIT labeling and DNA extraction, followed by sequencing to determine the alleles and mutation rates of somatic mutations in individual crucian carp. The F1 generation, which had a somatic bmp6a and bmp6b double mutation line with a somatic mutation rate of more than 95%, was selected as the parent for breeding to construct the F2 generation, and a homozygous bmp6a and bmp6b double mutation line was selected from the F2 generation. Among them, the knockout target sites of the bmp6 gene are shown in Table 1. Table 1 ； The sgRNA upstream primer shown in Table 1 and the sgRNA downstream primer with the sequence of 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3' were used for in vitro synthesis of sgRNA. The sgRNA in vitro amplification system was as follows: 2.5 mL each of 10 mM sgRNA upstream and downstream primers, 25 mL of 2× DreamTaq Master mix, and enzyme-free water to 50 mL. The sgRNA in vitro amplification program was denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and extension at 72°C for 5 min. After amplification, the PCR product was purified and recovered, and then sgRNA was transcribed in vitro using an RNA in vitro transcription kit. A 30 mL reaction system was established for each target site: 1 mg of sgRNA PCR recovered product, 10 mL of NTP Buffer Mix, and 2 mL of T7 RNA Polymerase Mix. mL, and add enzyme-free water to 30 mL; transcribe at 37℃ for 4 h. After the reaction, add 20 ml of enzyme-free water, mix well, and then add 2 ml of DNase I. Digest at 37℃ for 15 min to remove unreacted DNA.

2. the method for breeding carp strain without intermuscular spiny crucian carp according to claim 1, is characterized in that The microinjection method is: in vitro synthesized sgRNA and Cas9 protein are mixed at a molar concentration ratio of 3:1, incubated at room temperature for 10 minutes, and then 25% phenol red is added and injected into one-cell stage crucian carp embryos; among them, equal amounts of target site sgRNA on each exon are mixed and injected, the final concentration of each sgRNA is not less than 50ng / mL, and the injection volume per fertilized egg is 1nL±0.02nL.

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