[Effect of Different Caenorhabditis elegans U6 Promoters on the Efficiency of CRISPR/Cas9-Mediated Gene Editing].
Abstract — Entities Highlighted
Gene/ProteinChemicalDiseasePhenotypeAnatomyDev. Stage
To investigate the effects of Caenorhabditis elegans (C. elegans) endogenous U6 promoters on dpy-10 gene editing efficiency.
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 (Peft-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.
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 gRNAF+E scaffold did not show superior editing efficiency over the gRNA scaffold when paired with the optimal U6w05b2.8 promoter.
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.
Relation Network
Specialists Used
deepseek: 15
All Extracted Relations (15)
| Entity 1 | Relation | Entity 2 | Confidence | Specialist | Direction |
|---|---|---|---|---|---|
| U6 w05b2.8 | negatively regulates | gRNAF+E scaffold | 85% | deepseek | refutes |
| U6 r07e5.16 | positively regulates | dpy-10 | 85% | deepseek | supports |
| U6 w05b2.8 | positively regulates | dpy-10 | 85% | deepseek | supports |
| U6 c28a5.7 | positively regulates | dpy-10 | 85% | deepseek | supports |
| U6 f54c8.9 | positively regulates | dpy-10 | 85% | deepseek | supports |
| U6 k09b11.11 | positively regulates | dpy-10 | 85% | deepseek | supports |
| U6 r07e5.16 | negatively regulates | U6 w05b2.8 | 85% | deepseek | refutes |
| U6 k09b11.12 | negatively regulates | U6 w05b2.8 | 85% | deepseek | refutes |
| dpy-10 | causes phenotype | dpy-10 mutant phenotypes | 85% | deepseek | supports |
| CRISPR/Cas9 | positively regulates | dpy-10 | 85% | deepseek | supports |
| U6 promoters | positively regulates | CRISPR/Cas9 systems | 85% | deepseek | supports |
| U6 snRNA genes | expressed in | WormBase database | 85% | deepseek | supports |
| pSX524 plasmid | binding | Peft-3::cas9::tbb-2 terminator::U6 r07e5.16::dpy-10 sgRNA | 85% | deepseek | supports |
| microinjection protocol | therapeutic | C. elegans | 85% | deepseek | supports |
| U6 promoters | positively regulates | genome editing strategies | 85% | deepseek | supports |