Establishment Of The Pygo1 Gene Knockout Line Using CRISPR/Cas9 System And Its Preliminary Function Study

CRISPR(Clustered regularly interspaced short palindromic repeats)gene editing artifact,is considered a revolutionary technology in the field of genetics,in recent years,the rapid development of this technology and soon swept the domestic and international scientific research community.CRISPR can be used to delete,add,activate or suppress target genes of other organisms,so it is a very accurate genetic weapon for gene knockout and knockin.Zebrafish as a classic experimental model animals have their own advantages,and they are used in genetics and molecular biology and other research areas.Therefore,this article selected CRISPR targeting experiment in the zebrafish body.The pygo1 gene is a candidate gene for human cardiac development that has been identified in the laboratory,at present,there are no reports of mutations in the gene and related functions.To explore the biological function of the gene in cardiac development,in this paper,the construction of zebrafish pygo1 gene mutations was carried out by CRISPR / Cas9 gene knockout technique.Firstly,the functional domain of pygo1 gene was analyzed by some specialized websites,and four g RNAs with goodspecificity were designed between exon 1,exon 1 and intron 2,respectively.And the four g RNAs were transcribed and synthesized.Then g RNA and h Cas9 were injected into zebrafish embryos in a cell phase,respectively,after 48 hours of culture,the embryonic genome was collected for validity analysis,the embryos with validness were cultured for three months and then identified respectively,two F0 generation chimeras were successfully obtained.And then the screening of the two F0 generation chimera were heterosexual wild zebrafish hybridization to get a large number of offspring,raisedto three months after further screening identification.After a series of screening and identification work,a F1 hybrids with gene mutations were selected.And then continue to F1 heterozygous gene mutation in the same male and female individuals to get F2 homozygous.At present,the stable knockout model of 4bp and7 bp deletion was obtained.Which laid the foundation for the study of the biological function of the gene and the role played by the development of zebrafish pygo1 gene knockout stability.