The application of somatic CRISPR‐Cas9 to conditional genome editing in Caenorhabditis elegans

Abstract

Summary: Forward and reverse genetic approaches have been well developed in the nematode Caenorhabditis elegans; however, efficient genetic tools to generate conditional gene mutations are still in high demand. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) system for genome modification has provided an additional tool for C. elegans researchers to achieve simple and efficient conditional targeted mutagenesis. Here, we review recent advances in the somatic expression of Cas9 endonuclease for conditional gene editing. We present some practical considerations for improving the efficiency and reducing the off-target effects of somatic CRISPR-Cas9 and highlight a strategy to analyze somatic mutation at single-cell resolution. Finally, we outline future applications and consider challenges for this emerging genome editing platform that will need to be addressed in the future. genesis 54:170-181,

Neuronal IL-17 controls Caenorhabditis elegans developmental diapause through CEP-1/p53

“…The ilc-17.1 gene has not been identified in previous genetic screens for dauer pathway components ( 3 , 5 , 30 – 32 ). Therefore, although no off-target effects using CRISPR have been observed in C. elegans even after whole genome sequencing ( 33 – 35 ), we conducted additional experiments to confirm the role of ilc-17.1 in dauer entry by i) examining the developmental phenotype of another independently generated CRISPR deletion in ilc-17.1, syb5297 (loss of 2188 bp of 2980 bp; SI Appendix , Fig. S1 A ), ii) backcrossing ilc-17.1 (syb5296) mutants, and iii) down-regulating ilc-17.1 and its receptor ( 26 , 27 ) in a wild-type (N2) background using RNA interference (RNAi) and auxin-induced degradation (AID).…”

Neuronal IL-17 controlsC. elegansdevelopmental diapause through CEP-1/p53

“…Ilc-17.1 has not been identified in previous genetic screens for dauer pathway genes(3, 5, 30–32). Therefore, although no off-target effects using CRISPR have been observed in C. elegans even after whole genome sequencing (33–35), we conducted additional experiments to confirm the role of ilc-17.1 in dauer entry, by (i) examining the developmental phenotype of another independently generated CRISPR deletion in ilc-17.1, syb5297 (loss of 2188 bp of 2980 bp; Supplementary Fig. S1a), (ii) backcrossing ilc-17.1 (syb5296) mutants, and (iii) downregulating ilc-17.1 and its receptor(26, 27) in a wild-type (N2) background using RNA interference (RNAi) and auxin-induced degradation (AID).…”

Loss-of-function genetic tools for animal models: cross-species and cross-platform differences

“…This can be overcome by transgenic expression of Cas9, as has been done in mice 86 , zebrafish 73 and C. elegans 87 , and so only necessitating the delivery of gRNAs and, if required, repair templates. Alternatively, to avoid the difficulties of Cas9 and gRNA delivery, CRISPR can be applied in cultured cells, which are then injected into mice, allowing genome-wide in vivo screens 88,89 .…”