ASAP1 (Arf-GAP with SH3 domain, the ankyrin repeat and the PH domain) is the GTPase activating protein of the small G protein Arf. To understand more about the physiological functions of ASAP1 in vivo, we chose to use the zebrafish as an animal model, and analyzed the characterization of asap1 using loss-of-function studies. Here, two isoforms in zebrafish, asap1a and asap1b, were found to be homologous to human ASAP1, and the gene knockout zebrafish lines for asap1a and asap1b were established using the CRISPR/Cas9 technique with different insertions and deletions of bases. Zebrafish with asap1a and asap1b co-knockout showed a significant reduction in survival and hatching rates, as well as an increase in malformation rates during the early stages of development, while the asap1a or asap1b single knockout mutants did not affect the growth and development of individual zebrafish. Exploring the gene expression compensation between asap1a and asap1b using qRT-PCR, we found that asap1b had increased expression when asap1a was knocked out, showing a clear compensatory effect against asap1a knockout; In turn, asap1a did not have detectable compensating expression after asap1b knockout. Furthermore, the co-knockout homozygous mutants displayed impaired neutrophil migration to Mycobacterium marinum infection, and showed an increased bacterial load. Together, these are the first inherited asap1a and/or asap1b mutant zebrafish lines by the CRISPR/Cas9 gene editing approach, and by serving as useful models, they can significantly contribute to better annotation and follow-up physiological studies of human ASAP1.

ASAP1（带有 SH3 结构域、锚蛋白重复序列​​和 PH 结构域的 Arf-GAP）是小 G 蛋白 Arf 的 GTP 激活蛋白。为了更多地了解ASAP1在体内的生理功能，我们选择使用斑马鱼作为动物模型，并通过功能丧失研究来分析ASAP1的表征。本研究发现斑马鱼中的两个亚型asap1a和asap1b与人类ASAP1同源，并利用CRISPR/Cas9技术通过不同的碱基插入和缺失建立了asap1a和asap1b基因敲除斑马鱼品系。asap1a和asap1b共敲除的斑马鱼在发育早期表现出存活率和孵化率显着降低，畸形率增加，而asap1a或asap1b单敲除突变体并不影响个体的生长和发育斑马鱼。利用qRT-PCR探讨asap1a和asap1b之间的基因表达补偿，我们发现当asap1a被敲除时， asap1b的表达增加，对asap1a敲除表现出明显的补偿作用；反过来，asap1b敲除后， asap1a没有可检测到的补偿表达。此外，共敲除纯合突变体显示中性粒细胞向海分枝杆菌感染的迁移受损，并且细菌负荷增加。总之，这些是第一个通过 CRISPR/Cas9 基因编辑方法遗传的asap1a和/或asap1b突变斑马鱼品系，通过作为有用的模型，它们可以为人类 ASAP1 的更好注释和后续生理研究做出重大贡献。