Abstract: The ADGRF3A protein, encoded by the adgrf3a gene, is a member of the family of adhesion G protein-coupled receptors (aGPCRs) and is mainly expressed in embryos at 5 days after fertilization and in the head and gonads of adult zebrafish. It contains a GPS proteolytic site and seven transmembrane structural domains that interact with G proteins to perform its function. Zebrafish is a model organism for studying physiology and pathology during early development and adulthood. In order to study the role of adgrf3a in early zebrafish development, an adgrf3a knockout zebrafish line was constructed using CRISPR-Cas9 technology. Firstly, the knockout site of the gene was screened out by using analyzing software, and the guide DNA (sgDNA) was amplified by using polymerase chain reaction, next the guide RNA (sgRNA) was obtained by in vitro transcription and then the sgRNA and the Cas9 protein were co-injected into the zebrafish 1-cell-stage embryos. Then, the effectiveness of gene editing was tested, and the results showed that the injected embryos had base deletions, indicating that the gene knockout was successful. The F1 generation of zebrafish resulting from crosses between chimeras and wild-type zebrafish were genotyped, screened for adgrf3a mutant heterozygotes, and their adgrf3a mutant alleles were subjected to Sanger sequencing to determine the establishment of adgrf3a knockout lines. Subsequently, the adgrf3a mutant heterozygous zebrafish were self-crossed to obtain adgrf3a mutant homozygous zebrafish. As observed and imaged by stereomicroscopy, the adgrf3a mutant zebrafish did not show a phenotype significantly different from that of the wild type. However, whether the development of tissues and organs in vivo is altered needs to be further verified. The establishment of this knockout line lays the foundation for exploring the role of adgrf3a in early development and pathology.
Key words: zebrafish; adgrf3a; CRISPR-Cas9; gene knockout; gene heterozygous mutant strain
（Acta Laser Biology Sinica, 2023, 32(5): 423-430）