Fig. 2

C. briggsae-specific gRNA induces targeted recombination in the hybrid F1 progeny between C. briggsae and C. nigoni but not in the introgression strains. A Schematic diagram showing the generation and validation of targeted recombination on chromosome IV. Specifically, a double strand break is induced by the injection of Cas9 and a C. briggsae-specific gRNA into the females of an introgression strain (ZZY10458) or of the F1 hybrids between ZZY0734 male and C. nigoni female. The right arm of the C. briggsae chromosome IV is marked with a GFP insertion. The F1 hybrid or the introgression females are backcrossed to C. nigoni wild isolate males. The crossing progeny are screened for the presence of targeted recombinant by single-worm PCR (swPCR) amplification of the C. briggsae-specific genomic fragments flanking the expected target site. The absence ( −) and presence ( +) of a PCR product on the left and right side of the target site indicate a successful targeted recombination. Simultaneous presence or absence of PCR product(s) on both sides of the target site indicates no targeted recombination. For simplicity, only the chromosome IV is shown. B Targeted recombinant is absent in the crossing progeny between the introgression strain and C. nigoni (top left) but is present in crossing progeny between the hybrid F1 and C. nigoni (bottom left) using the C. briggsae-specific gRNA targeting gene CBG23872. Shown on the right is the bar plot of targeted recombinant frequency with the total number of screened worms (n) indicated. Error bar represents 95% confidence interval calculated with the Agresti-Coull method. (****) p < 0.0001 (chi-square test). C Validation of the targeted recombinants in the hybrid F1 progeny (B) through sequencing the recombination boundaries. Top: the sequence alignment (pink block) flanking the gRNA target site. Bottom: sequencing results for the boundaries of three independent targeted recombinants