English: "(A) Cas9, D10A, and H840A nucleases expressed under the control of vasa promoter from three equivalent insertions (in X chromosome locus yellow) were used to induce allelic repair in yccw wATG− CR+/yvasaCas9 wATG+ CS1−, yccw wATG− CR+/yvasaD10A wATG+ CS1−, and yccw wATG− CR+/yvasaH840A wATG+ CS1− females. Control animals were yccw wATG− CR+/y+ wATG+ CS1. Left panels show typical repair phenotypes, and diagrams on the right represent cutting or nicking by each different nuclease. (B) Allelic repair was quantified by image analysis using ImageJ. Nickases are more efficient than Cas9 in eliciting HTR, and H840A is more efficient than D10A for repairing the CS1− allele. (C) Quantitative analysis of HTR of the CS1− allele by deep sequencing. Correction percentages after adjustments (see Materials and Methods) from five independent reads were plotted for each genotype (no nuclease control CR+/CS1−, +Cas9, +D10A, and +H840A). Results confirm the trend calculated using pigment and Sanger sequencing analysis: Repair by D10A is significantly higher (41%) than by Cas9 (27%). H840-elicited repair (51%) is significantly more efficient than by D10A. ***P < 0.001 and *P < 0.05. (D) Pie chart representation of deep sequencing analysis. Color coding—pink, donor CR+ alleles; white, intact CS1− alleles; dark purple, NHEJ mutations (centered at cut site); gray, PCR-induced recombination #1 and some asymmetrical HTR and NHEJ events (only for Cas9, D10A, and H840 samples; see fig. S9); light blue sectors, PCR-induced recombination #2; light purple sectors, PCR-induced substitutions. This representation allows global visualization of different categories of events following Cas9-, D10A-, and H840-dependent cleavage in wATG− CR+/y+ wATG+ CS1− individuals. Nickases are more effective at producing HTR than Cas9, with H840 inducing the highest levels of conversion. Cas9 induces high levels of NHEJ events, while D10A and H840 only elicit low levels of NHEJ and leave ~18 to 23% of intact CS1− alleles."