Fig. 1.

LbCas12a mediates efficient temperature-sensitive gene editing in Drosophila. (A) Schematic of the Cas12a genome engineering system. Cas12a possesses RNase activity and can cleave crRNA arrays into individual crRNAs. Upon on-target binding, Cas12a cuts dsDNA, producing DSBs with 5′ overhangs. These are often imprecisely repaired by the endogenous DNA repair machinery, resulting in mutations at the target locus. (B) Schematic of the workflow of experiments presented in C and D. Single Cas12a crRNAs are cloned into expression vectors pCFD7 (for AsCas12a) or pCFD8 (for LbCas12a). Plasmids are integrated at defined genomic locations and crossed to the respective nuclease. Offspring inheriting Cas12a and crRNA transgenes are raised at different temperatures and assessed for on-target gene editing. (C) Cas12a-mediated mutagenesis of ebony (e) at different temperatures. Images of flies expressing crRNA-e and either AsCas12a or LbCas12a and raised at the indicated temperatures are shown. Disruption of e results in dark coloration of the cuticle. AsCas12a mediates efficient mutagenesis only at 29 °C, while LbCas12a mediates gene disruption at all tested temperatures. (D) Assessment of gene-editing efficiency of 11 target sites by Amp-seq. Activity of each nuclease was tested at 18 °C, 25 °C, and 29 °C. Genomic target sites of each crRNA were PCR amplified and amplicon pools were subjected to deep sequencing. Gene-editing activity was strongly temperature-sensitive with both nucleases. LbCas12 displayed much more robust activity, mediating strong mutagenesis with 7 of 11 crRNAs at 29 °C, compared to 2 of 11 in the case of AsCas12a. Rates of modified reads being called due to sequencing errors was between 0.5% and 1.6% (SI Appendix, Fig. S1).