Generation Of Titin-Cap Gene Knockout Lines Using CRISPR/Cas9 System And A Preliminary Study On Its Function

The occurrence of heart disease is regulated by multiple genes,Study of cardiac development-related regulatory genes is a breakthrough in understanding the mechanism of heart disease,but also an important means of prevention and treatment of such diseases.Gene knockout and knocking are commonly used tools for studying gene function,CRISPR(Clustered regularly interspaced short palindromic repeats)system has been widely used in gene research of various models,and zebrafish is a model Animals have unique characteristics in the fields of genetics and molecular biology,our laboratory has a perfect zebrafish model biological research platform.Tcap gene is a candidate gene for human cardiac development which has been identified in our laboratory.And at present,there are no reports on the establishment and related mechanism of mutant lines.So,in order to explore the biological function of the gene in the development of the heart,this paper mainly used CRISPR/Cas9 gene knockout technique to construct the gene mutant strain of zebrafish.First,we analyzed the functional domain of the Tcap gene through the corresponding website and designed a specific g RNA with a specific exon region of the gene.And then the g RNA in vitro transcription synthesis,and then,we co-inject h Cas9 m RNA and g RNA into zebrafish embryos at one cell stage,after 48 hours,we collection the embryos for genomic PCR amplification,then send sequencing to determine the effectiveness of g RNA,the results showed that there were bimodal peaks in 5 F0 individuals.The TA clone sequence of these five fish was compared with the zebrafish wild-type WT sequence and found to have base deletion.In order to further study the mechanism of Tcap gene regulation of cardiac development,We cloned the CDS sequence of the zebrafish Tcap gene into p Tol2-CMLC2-IRES-EGFP vector to construct the overexpression plasmid of the zebrafish Tcap gene,and then co-injected the overexpression plasmid with the Tol2 transposase synthesized in vitro to the early embryo of zebrafish,to construct the overexpression of Tcap gene in zebrafish to study the effect of Tcap gene expression on zebrafish heart development in early embryos and to explore the role of Tcap gene in regulating cardiac development.Related research work is ongoing.In this paper,CRISPR/Cas9 gene knockout technique was used to construct knockout lines of zebrafish gene CXXC5 A and CXXC5 B gene,and the related research work was in progress.This study will lay the foundation for the further study of the molecular biological functions of Tcap,CXXC5 A and CXXC5 B genes in the whole embryo level,and provide a new research direction for clinical treatment.