Development and characterization of multiple highly efficient pgSIT systems. a Gender (♀, ♂, and ⚥) frequencies of trans-heterozygous F1 progeny resulting from crosses between double gRNAs (dsRNA) and Cas9 homozygous lines. Three dgRNAs, each targeting sxl, tra, or dsx combined with βTub, were bidirectionally crossed with three Cas9 lines driven by nanos (nos), vasa (vas), and Ubiquitin-63E (Ubi) promoters and were sufficient to ensure complete penetrance of both female lethality/masculinization and male sterility in each reciprocal cross (Supplementary Figs. , ). Gender frequencies and fertility in trans-heterozygotes were compared with those in the corresponding progeny of control crosses with Cas9 (bar groups to the left, solid lines) or dgRNAs (top panels, dashed lines) and wt flies. Bars represent means ± SD for three/four independent groups of parental flies. P>0.01**, P>0.001*** by a t test assuming unequal variance (black *) or, for male sterilization by Pearson’s chi-squared test for contingency tables (red *). b Order of targeted gene in the sex-determination pathway (top) and the corresponding knockout phenotype in progeny. Phenotypes of dgRNAs directed knockouts and intersex morphology in comparison to wt females and males. βTub, Sxl double-knockout females perish during pupal stages (Supplementary Fig. ). dgRNAβTub,Tra/+; nos-Cas9/+intersexes, but not dgRNAβTub,DsxF/+; nos-Cas9/+ intersexes, had sex combs, magnified inside inserts. c Variable expressivity of the number of sex comb bristles in βTub, Tra double-knockout intersexes. Scale bar shows 100 µm. d Internal reproductive organs in wt females: two ovaries (ov), seminal receptacle (sr), double spermatheca (sp), two accessory glands (ag), and uterus (ut). e Many dgRNAβTub,Tra/+; nos-Cas9/+ intersexes had one rudimentary ovary, and organs that resembled male accessory glands. f Many dgRNAβTub,DsxF/+; nos-Cas9/+ intersexes developed only a single ovary often times not connected with an oviduct and had organs that resembled male-specific accessory glands. g–h Male internal reproductive system in dgRNAβTub,Sxl/+; nos-Cas9/+ male. In comparison with wt testis i, elongated cysts with maturing spermatids were not found in the dgRNAβTub,Sxl/+; nos-Cas9/+ testis (h, i: arrows). Scale bars correspond to 500 µm