Effect of Caenorhabditis elegans U6 Promoters on CRISPR/Cas9-Mediated Gene Editing Efficiency

Objective This study investigated the effects of endogenous C. elegans U6 promoters on dpy-10 gene editing efficiency. Methods We screened endogenous U6 snRNA genes from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6 r07e5.16 promoter in pSX524 (P eft-3 :: cas9::tbb-2 terminator::U6 r07e5.16 :: dpy-10 sgRNA ) through molecular cloning. Following standardized microinjection protocols, we quantified gene editing efficiency and high-efficiency gene editing index based on dpy-10 mutant phenotypes in F1 progeny. Results Fifteen 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, k09b11.13 ) were identified from WormBase database. Comparative analysis revealed four U6 promoters ( w05b2.8, c28a5.7, f54c8.9 , and k09b11.11 ) significantly enhanced gene editing compared to other U6 promoters, including commonly used U6 r07e5.16 and U6 k09b11.12 promoters in C. elegans community. Notably, the gRNA F+E scaffold showed no improvement over the gRNA scaffold when paired with the optimal U6 w05b2.8 promoter. Conclusion This study identifies superior U6 promoters for C. elegans gene editing and demonstrates the critical role of promoter optimization in CRISPR systems, providing novel insights for technical refinement.