CRISPR/Cas9-mediated Knock-out Line Of The Asb5a Gene In Zebrafish

Asb5 is a member of the ASB family and may be involved in the development of arterial occlusive disease.Our previous study found that asb5 was specifically expressed in the human heart and skeletal muscle,and highly expressed in the heart,suggesting that it may be related to the development of the heart and skeletal muscle.But the biological function of the asb5 gene is currently unknown.The zebrafish has many advantages as a model organism,such as in vitro fertilization,embryo transparency,developmental observation under the microscope,homology with the human genome of up to 70%,and so on.In this study,zebrafish was used as a research model to construct the asb5 knock-out line,which provided the basis for the subsequent study of the function of the asb5 gene.In this study,knockout work was performed by designing a targeting site in each of the two exons,knocking out two target sites.This method is more specific than a single sgRNA mediated knockout and can result in the deletion of large fragments and is also easy to screen.Firstly,the basic biological information of asb5 gene was analyzed.The results showed that the homologous genes of human ASB5 gene in zebrafish were asb5a and asb5b.In this study,we selected the asb5a gene as a research object.First of all,one target site was designed on exons 2 and 3 of the gene and then sgRNA was synthesized in vitro and co-injected with Cas9 mRNA into cells of a single-cell stage embryo of wild-type zebrafish.After the embryo develops to 48 hours,the effectiveness of the target analysis,that is,the selection of a certain number of embryo extraction genome,PCR amplification test whether there is a mutation in the presence of the embryo,the preservation of target-effective embryos,feeding to sexual maturation,the FO-generation mutant screening,that is,by cutting the tail fin to extract the genome.After PCR amplification,the mutant strips were connected with the pMD18-T vector,and the plasmid was sequenced after transformation.The result of sequence comparison showed that there was a large segment deletion of asb5a gene in some fish of FO.The results show that the knockout of asb5a gene by CRISPR/Cas9 technology can cause effective gene mutation in zebrafish,which provides a condition for the functional study of asb5a gene.