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CN-120966918-B - Method for knocking out heteroploid changfeng crucian runx2b gene and application

CN120966918BCN 120966918 BCN120966918 BCN 120966918BCN-120966918-B

Abstract

The invention relates to the technical field of biology, in particular to a method for knocking out a heteroploid Changfeng crucian runx2b gene and application thereof. The invention designs gRNA-1 and gRNA-2 aiming at all alleles of runx2B of two sets of parents of carassius auratus gibelio and red carp of Xingguo, mixes two gRNAs transcribed and synthesized in vitro with Cas9 protein, and guides the mixture into animal poles of a single cell stage of fertilized eggs of the carassius auratus gibelio by adopting a microinjection mode, so that all 8 alleles of runx2B-A and runx2B-B can be knocked out efficiently. Any gRNA can guide the CRISPR/Cas9 system to edit 8 alleles at the same time, the gRNA-1 realizes 97.5% of synchronous knockout rate, the gRNA-2 realizes 95% of synchronous knockout efficiency, and when a double gRNA cooperative cutting strategy is adopted, the complete editing efficiency of 8 functional copies reaches 100%.

Inventors

  • LI XIAOHUI
  • LIANG HONGWEI
  • GENG ZIYANG

Assignees

  • 中国水产科学研究院长江水产研究所

Dates

Publication Date
20260512
Application Date
20250804

Claims (10)

  1. 1. A method for knocking out a heteroploid changium changii runx2b gene, which is characterized by comprising the following steps: S1, designing gRNA-1 and gRNA-2 aiming at two sets of runx2b alleles of parent sources in the Changfeng crucian carp, wherein the gRNA-1 or the gRNA-2 simultaneously specifically targets 8 alleles of runx2b, the sequence of a target site of gRNA-1 is shown as SEQ ID NO. 1, and the sequence of a target site of gRNA-2 is shown as SEQ ID NO. 2; S2, performing overlap PCR amplification and purification by using a conserved downstream Scaffold overlap primer and an upstream primer with a T7 promoter and specifically containing a gRNA-1 target site sequence or a gRNA-2 target site sequence, performing in vitro transcription and purification by using the purified PCR product as a template to obtain gRNA-1 and gRNA-2 respectively, mixing the gRNA-1, the gRNA-2 and Cas9 proteins, and microinjecting the mixture into fertilized eggs of the Changfeng crucian carp in a single cell stage.
  2. 2. The method of claim 1, wherein in step S2, after mixing the gRNA-1, the gRNA-2, and the Cas9 protein, the final concentration of both the gRNA-1 and the gRNA-2 is 400 ng/μl and the final concentration of the Cas9 protein is 5 μΜ.
  3. 3. The method of claim 2, wherein in step S2, 0.005 μl of the mixture of gRNA-1, gRNA-2 and Cas9 protein is injected per 1 fertilized egg.
  4. 4. The method according to claim 1, wherein in the step S2, fertilized eggs of the Changfeng crucian carp are obtained by a gynogenesis operation before microinjection, wherein the gynogenesis operation is to inject an oxytocic into the female Changfeng crucian carp and the male Xingguo red carp respectively, wherein the oxytocic injection dose of the Xingguo red carp is half of that of the Changfeng crucian carp, and artificial insemination is performed to obtain fertilized eggs after the Changfeng crucian carp begins spawning.
  5. 5. The method of claim 1, further comprising the steps of incubating fertilized eggs of the single cell stage of the long-chain crucian carp after injection to the juvenile stage, taking tail fins, extracting genomic DNA, performing PCR amplification by using SEQ ID NO. 6-13 as a primer, sequencing PCR products, and selecting the long-chain crucian carp with the run 2B-A and run 2B-B genes from the carassius auratus gibelio source and the red carp of Xingguo being knocked out simultaneously.
  6. 6. The method of claim 1, wherein in step S2, the upstream primer for the overlap PCR amplification of gRNA-1 is shown as SEQ ID NO.3, the downstream primer is shown as SEQ ID NO.5, and the upstream primer for the overlap PCR amplification of gRNA-2 is shown as SEQ ID NO.4, the downstream primer is shown as SEQ ID NO. 5.
  7. 7. The gRNA combination of the heteroploid Changfeng crucian carp runx2b gene is characterized by comprising gRNA-1 and gRNA-2, wherein the target site sequence of the gRNA-1 is shown as SEQ ID NO. 1, and the target site sequence of the gRNA-2 is shown as SEQ ID NO. 2.
  8. 8. Use of the gRNA combination of claim 7 for breeding a mutated strain of changfu crucian carp.
  9. 9. A knockout composition of the heteroploid changium runx2b gene comprising the gRNA combination of claim 7 and a Cas9 protein.
  10. 10. A knockout kit for the run 2b gene of heteroploid changium crucian carp, comprising the gRNA combination of claim 7 and a Cas9 protein.

Description

Method for knocking out heteroploid changfeng crucian runx2b gene and application Technical Field The invention relates to the technical field of biology, in particular to a method for knocking out a heteroploid Changfeng crucian runx2b gene and application thereof. Background Crucian carp (Carassius auratus) is an important freshwater aquaculture fish in China, the 2023-year aquaculture yield reaches 284.03 ten thousand tons, and the 5 th freshwater aquaculture yield makes an important contribution for guaranteeing stable and effective supply of aquatic products (Chinese fishery statistics annual survey, 2024). The Changfeng crucian carp is a new crucian carp variety which is bred by a system which is started and bred by the aquatic institute of China aquatic science research institute and the aquatic organism institute of China academy of sciences in 2008, and the 6 th generation heterogenic gynogenesis is completed in 2015, and the new crucian carp variety is formally inspected and approved by the new national aquatic product variety in 2016. The Changfeng crucian carp uses a carassius auratus gibelio (Carassius auratus gibelio) D line as a female parent and a carp moving core fish Xingguohong carp line (Cyprinus carpio var. Xingguonensis) as a male parent, exogenous genetic materials are integrated through a heterogenic gynogenesis technology, the genome of the carassius auratus gibelio comprises a female parent heterogenic carassius auratus gibelio whole genome and a male parent Xingguohong carp whole genome, a genetically stable heterogenic octaploid group is formed, and the chromosome number is about 208, compared with that of the heterogenic carassius auratus gibelio, about 50 chromosomes are more. The growth rate of the Changfeng crucian carp is fast, the growth rates of 1-year-old fish and 2-year-old fish are respectively improved by more than 25.0% and more than 16.0% compared with that of common silver crucian carp, the meat quality is fine, the muscle fiber density is only 184+/-24 pieces per square millimeter, the muscle fiber density is 23% -37% thinner than that of common silver crucian carp, the taste is better, the nutrition is rich, the DHA content is 10.3% and is 2.28 times that of Pengjean carp, the arachidonic acid content is 8.3% and is 1.62 times that of Pengjean carp, in 21 fatty acid detection, the unsaturated fatty acid and DHA content are improved by 115.16% and 255.17% compared with that of the heterogeneous silver crucian carp D system, the stress resistance is strong, and the method is suitable for various national controllable fresh water bodies (such as ponds, paddy fields and the like). Runt-related transcription factor (Runx 2) is a core member of the Runx transcription factor family, playing a key role in the skeletal development of vertebrates. Studies show that there are two direct homologous genes Runx2a and Runx2b in the Runx2 gene in zebra fish, wherein Runx2b is proved to be a core transcription factor for regulating fish intramuscular thorn formation. Guan Ningnan and the like find that runx2b has obvious up-regulation characteristics in the differentiation process of osteoblasts by comparing the gene expression difference of the megalobrama amblycephala bone tissue cell line and the muscle cell line, and provide important basis for the molecular mechanism research of the formation of intramuscular thorns. Nie Chungong and the like further construct a runx2b deletion zebra fish model through a gene editing technology, prove that the gene deletion can cause complete deletion of the myothorns and does not influence other skeletal development, and the functional localization of the runx2b serving as a key gene for the myothorn development is defined. The gene editing technology is used as an effective means for precisely modifying target genes, and the development of the gene editing technology is subjected to homologous recombination, zinc Finger Nucleases (ZFNs), effector nucleases (TALENs) and other stages. Among them, the CRISPR/Cas9 system has become the current mainstream technology because of its advantages of high editing efficiency and strong specificity. However, the multiplication of the number of the medium genes in the genome of the changium octopus, which leads to the remarkable enhancement of the gene redundancy effect, the knockout of the carassius auratus gibelio or the knockout of the gene sequence from the red carp of the Xingguo alone cannot effectively reduce the number of the intramuscular thorns, and the low efficiency of gene editing and targeting often leads to the phenotype that is randomly distributed in the filial generation and the proportion is uncontrollable. A large number of repeated sequences in the genome form a complex secondary structure, the effective combination of the gRNA and a target site is interfered, and the existence of paralogous sequence variation causes nonspecific cutting and the off-target risk is multiplied. After DN