Construction Of The Zebrafish Cxxc5a Gene- Knockout Line Using CRISPR/Cas9

Objective: The CXXC5 gene is widely expressed and evolutionarily conserved in the heart and has been shown to affect cardiomyocyte proliferation and cardiac cyclization in the early stages of zebrafish development.Gene editing technology is a precise and efficient genetic engineering method,it can edit the target genes in organisms by inserting,knocking,modifying or replacing specific DNA fragments.CRISPR/Cas9 is the most dazzling and promising genetic editing technology in the current field of life sciences.In this study,the knockout strain of zebrafish cxxc5 a gene was constructed by CRISPR/Cas9 gene editing technology,in order to further explore the specific mechanism of cxxc5 a gene affecting early heart development.Methods: The complete sequence of cxxc5 a gene and other related information are obtained by online analysis,and two higher-scoring target sites and target identification primers are designed on the exon 2 of cxxc5 a gene by bioinformatics software,"double target site knockout" method are performed to knockout the gene.(1)Construction of targeting vector: separately synthesize gRNA primers and identification primers for targeting,through a series of operations such as PCR amplification,agarose gel electrophoresis,DNA recovery and purification,in vitro transcription,etc.,finally obtain high-purity gRNA and hCas9 mRNA for targeting.(2)Microinjection: The constructed high-purity targeting gRNA and hCas9 mRNA are mixed in a certain ratio,and then be injected into the wild zebrafish-cell stage embryo by microinjection.(3)Detection and analysis of knockout effect: F0 embryos after injection are cultured for 72 hours,and a certain number of embryos are randomly selected for genome extraction,a series of work such as PCR amplification,agarose gel electrophoresis,DNA recovery and purification,ligation of pMD18-T vector,transformation,cloning,plasmid extraction,sequencing,and sequence alignment are used for target effectiveness analysis.After the remaining embryos are cultured for 4 months to sexual maturity,the tails of adult F0 generation zebrafish genome are cut down for genome extraction,the experimental procedure involved in the above-mentioned targeting effectiveness analysis is repeated,and finally the zebrafish F0 generation chimeric mutant which effectively knocked out the cxxc5 a gene is screened and obtained.(4)Screening and study of F1 generation knockout zebrafish: the above F0 generation chimeric mutant is hybridized with wild type zebrafish,and the obtained F1 generation zebrafish embryo is detected and analyzed;after 72 h of culture,a certain number of embryonic genomes are randomly selected,it is preliminarily identified by PCR amplification and agarose gel electrophoresis whether the cxxc5 a gene knockout of F0 generation zebrafish is effectively inherited to F1 generation;continue to culture the remaining F1 embryos to 5 months of sexual maturation,and then cut the tail to extract the genome and analyze.The zebrafish F1 hybrid mutant that knocked out the cxxc5 a gene,which is stably inherited,is finally screened.Results: High purity target gRNA and hCas9 mRNA were successfully synthesized in vitro;After microinjection,the target product band was generated by gel electrophoresis of PCR product for F0 generation embryo targeting analysis.Further sequencing and sequence alignment revealed that the F0 generation embryonic cxxc5 a gene deleted the base sequence between the two target sites,and the expected gene editing occurred in the embryo;the target product band was generated by gel electrophoresis of PCR products screened by F0 generation adult fish chimeric mutants.Further sequencing and sequence alignment showed that the cxxc5 a gene of some F0 generation adult zebrafish individuals had a frameshift deletion mutation,and the F0 generation chimeric mutant was successfully obtained.Selected appropriate F1 embryos,and analyzed and identified whether the knockout strain is inherited to F1 generation,but the gel electrophoresis results of the PCR products showed that the corresponding target bands were not present;after F1 embryos were cultured to sexual maturity,the gene knockout F1 hybrid mutants were continuously screened.In the zebrafish F1 generation that has been screened,the gel electrophoresis results of the PCR products also showed no target bands,and the reason is still under analysis.Screening for the remaining F1 generations is also underway.It is not yet certain whether the F0 gene knockout has been stably inherited to the F1 generation.Conclusion: This study used CRISPR/Cas9 gene editing technology to efficiently edit the cxxc5 a gene of zebrafish,which resulted in a frameshift deletion mutation.The cxxc5 a gene was knocked out at the overall level of the embryo,and a chimeric mutant was obtained,which laid an important foundation for the construction of the subsequent gene knockout homozygous lines.