Figure 3.

Resistance alleles accumulates over subsequent generations and restricts homing. Resistance alleles are expected to be immune to the further cleavage by the same Cas9/gRNA system and if their carrier is fertile can propagate at the expense of homing. (A) To explore this phenomenon, F₂ ♀ #5 and F₂ ♀ #6 collected among progeny of F₁ ♀ #2 were genetically crossed with w– and w+ males, respectively, and their F₃ progeny were scored. While the cleavage rate in F₂ germ cells decreased only in F₂ ♀ #6 with Ubi-Cas9 (red arrow) likely due to the rise of functional w^R1 alleles, the homing frequency fell significantly for each tested split-drive system with and without Cas9 gene (green arrows). The fall of homing rate was accompanied by the accumulation of the w^R2 alleles. (B) Accumulation of w^R2 alleles resistant to cleavage by Cas9/gRNA^w restricted homing of GDe. Frequencies of homing and resistance alleles were averaged for all tested promoters and presented separately for progeny of heterozygous and trans-heterozygous females, F₂ ♀ #5 and F₂ ♀ #6, respectively. Resistance allele frequency increased from 28.5% or 19.9% to 92.6% or 82.6%, respectively, between F₂ and F₃ (blue arrows) and caused the dramatic decline in homing from 69.0% or 73.0% to 6.1% or 9.2%, respectively (green arrows). Notably, scoring of w^R2 alleles in w– recessive background resulted in the higher estimation of white LOF mutations alleles, since w^R2 alleles were complemented by w+ alleles inherited from wild type males. Bar plots show the average ± SD over at least three biological replicate crosses. Statistical significance was estimated using a t-test with equal variance. (P ≥ 0.05^ns, P < 0.05*, P < 0.01**, and P < 0.001***).