Abstract: Heart and neural crest derivatives expressed transcription factor 2 (Hand2) is a member of bHLH family, also known as dHand, Hed and Thing2, which can be expressed in human, mouse, chicken, zebrafish, Drosophila and other model animals. Domestic and foreign studies have shown that Hand2 mainly affects the development of the right ventricle and plays a key role in the maintenance of right ventricular progenitor cells and morphogenesis of the right ventricle. In order to further study the mechanism of Hand2 gene involved in heart development, a zebrafish Hand2 gene knockout lines was constructed by CRISPR/Cas9 gene editing technology. According to the analysis of bioinformation website, the optimal two target sites were screened, and primers were designed and synthesized. The cDNA of the Hand2 gene sgRNA was obtained by PCR amplification, and then transcribed into mRNA. The mRNA of the two target sites sgRNA and Cas9 protein were co-injected into the Ⅰ-cell stage of zebrafish embryos. The effectiveness of gene knockout was tested in zebrafish embryos and adults, and it was found that there were base deletions and insertions near the target site, indicating that CRISPR/Cas9 was effective for zebrafish Hand2 knockout. In this study, a knockout model of Hand2 gene in zebrafish was constructed using CRISPR/Cas9 gene editing technology, which laid a foundation for the later study of the mechanism of Hand2 gene in human diseases and the clinical development of novel therapeutic targets.
Key words: zebrafish; Hand2 gene; CRISPR/Cas9; gene-knockout; therapeutic targets
（Acta Laser Biology Sinica, 2022, 31(2): 142-148）