Objective To investigate the effects of Caenorhabditis elegans (C. elegans) endogenous U6 promoters on dpy-10 gene editing efficiency.

Methods We screened endogenous U6 small nuclear RNA (snRNA) genes of C. elegans from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6_r07e5.16 promoter in the pSX524 plasmid (P_eft-3::cas9::tbb-2 terminator::U6 _r07e5.16::dpy-10 sgRNA) through molecular cloning. Gene editing was performed in wild-type C. elegans using a standardized microinjection protocol. Gene editing efficiency and the high-efficiency gene editing index were quantified based on the screening of dpy-10 mutant phenotypes in the F1 progeny.

Results A total of 15 U6 snRNA genes (r07e5.16, f35c11.9, t20d3.13, k09b11.15, k09b11.16, w05b2.8, c28a5.7, f54c8.9, k09b11.11, k09b11.12, k09b11.14, t20d3.12, f54c8.8, f54c8.10, and k09b11.13) were identified from the WormBase database. Based on the editing efficiency and high-efficiency gene editing index, the activity of these promoters was evaluated, and 4 U6 promoters (w05b2.8, c28a5.7, f54c8.9, and k09b11.11) were found to have significantly enhanced gene editing success rates, outperforming other promoters, including U6_r07e5.16 and U6_k09b11.12, which are commonly used in the C. elegans research community. Notably, the gRNA^F+E scaffold did not show superior editing efficiency over the gRNA scaffold when paired with the optimal U6_w05b2.8 promoter.

Conclusion In this study, U6 promoters that significantly improve gene editing efficiency in C. elegans are identified and the critical role of promoter optimization in CRISPR-Cas9 systems is highlighted. These findings provide a valuable foundation for improving genome editing strategies and offer new ideas for optimizing the CRISPR technology applied in nematode research.