Precision guided sterile insect technique (pgSIT), an assessment of gene targets with single guide RNAs (sgRNAs). a A schematic of pgSIT utilizing two components of the binary CRISPR/Cas9 system, Cas9 and gRNAs, maintained as separated homozygous lines, their cross results in simultaneous knockouts of a gene required for female viability and a gene required for male fertility resulting in survival of only F₁ sterile males. b A schematic of sex-specific alternative splicing in sxl, tra, and dsx regulated by female expression of Sxl and Tra proteins (gray lines) (modified from ref. ). Disruption of female-specific exons of key sex-determination genes, sxl, tra, and dsx, disrupts female development. PgSIT exon targets indicated by yellow crosses. c Bar graphs of average gender frequencies in F₁ progeny. Two top panels depict gender frequencies from bidirectional control crosses of homozygous sgRNA lines to wild type (wt), indicating that both fertile females and males (♀ and ♂) are present at similar ratios, but no sterile intersexes (⚥) were identified. Bottom two panels show gender frequencies from crosses of homozygous nanos-Cas9 (nos-Cas9) to wt (control) and four homozygous sgRNA lines (experiment). Independent of maternal or paternal Cas9 inheritance, 100% of trans-heterozygous sgRNA^Sxl females were lethal, 100% of trans-heterozygous sgRNA^Tra and sgRNA^DsxF females were masculinized into sterile intersexes (⚥), and 100% of trans-heterozygous sgRNA^βTu males were sterile. Gender frequencies and fertility in trans-heterozygotes were compared to those in the corresponding progeny of control crosses with nos-Cas9 (solid lines) or sgRNAs (dashed lines) and wt flies. Bars represent means ± SD for three/four independent groups of parental flies. 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 *)

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. ). dgRNA^βTub,Tra/+; nos-Cas9/+intersexes, but not dgRNA^βTub,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 dgRNA^βTub,Tra/+; nos-Cas9/+ intersexes had one rudimentary ovary, and organs that resembled male accessory glands. f Many dgRNA^βTub,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 dgRNA^βTub,Sxl/+; nos-Cas9/+ male. In comparison with wt testis i, elongated cysts with maturing spermatids were not found in the dgRNA^βTub,Sxl/+; nos-Cas9/+ testis (h, i: arrows). Scale bars correspond to 500 µm

Zygotic expression of CRISPR/Cas9 components ensures 100% penetrance. a Genetic quantification of dominant effect by maternal loading of Cas9 protein. Genotypes, gender frequencies, and fertility of progeny flies generated by reciprocal crosses between homozygous dgRNAs and heterozygous Cas9 flies. Bars represent means ± SD for three/four independent groups of parental flies. Gender frequencies from crosses with heterozygous maternal Cas9 were compared with those from the corresponding crosses with heterozygous paternal Cas9. P > 0.01**, P > 0.001*** by a t test assuming unequal variance. Solid bars indicate inheritance of Cas9 as a gene, while striped bars indicate inheritance of + allele. b Combinations of genotypes and maternal/zygotic contributions in embryos, and their penetrance. c Accumulation of high levels of biallelic mosaicism (BM) throughout development leads to the loss of gene function at the organismic level and ensures complete penetrance of induced phenotypes: lethality (lethal biallelic mosaicism (LBM)), female masculinization, or male sterility. Complementation of gene function in some cells by uncleaved wt alleles, and resistance alleles generated by NHEJ, are not sufficient to rescue the induced phenotype at the organismic level and therefore 100% of trans-heterozygous progeny have the induced phenotypes. Boxes get smaller and more abundant as cells divide

Competitiveness and longevity of pgSIT males and modeling data. a An experimental setup to estimate the mating competitiveness of dgRNA^βTub,Sxl/+; nos-Cas9/+ sterile males (marked with red) competing against wt males to secure matings with wt females. A mated female is resistant to the next mating for around 24 h^,, and the mating success of sterile males was evaluated by fertility decrease (aka. increase of unhatched egg rate). b Bars graph percentages of laid and hatched eggs (Supplemental Table ). The presence of one sterile male resulted in a significant decrease in female fertility (#3 vs. #2) that could not be accounted by removal of one wt male (#2 vs. #1). Bars represent means ± SD for five replicates. #3 to #2 and #1 P > 0.003**, P > 0.0001*** by a t test assuming unequal variance. c Survival curves of wt males (blue line) and two types of dgRNA^βTub,Sxl/+; nos-Cas9/+ sterile males, with paternal (red line) or maternal (green line) Cas9 inheritance. Survival curves show nonparametric maximum likelihood estimates for three male groups, along with 95% confidence intervals in light shade and dark shade for representational nonuniqueness. The y-axis shows the estimated survival percentage. Both types of pgSIT males lived significantly longer than wt males (P < 2.2^–16), while no significant difference was found between two types of pgSIT males by Sun’s generalization of the log-rank test. d Model-predicted impact of releases of pgSIT eggs on Aedes aegypti mosquito population density with comparison to releases of Wolbachia-based incompatible insect technique (IIT), release of insects carrying a dominant lethal gene (RIDL), and female-specific RIDL (fsRIDL). Releases are carried out weekly over a 6-month period with release ratios (relative to wild adults) shown in the key. Model predictions were computed using 2000 realizations of the stochastic implementation of the MGDrivE simulation framework for a randomly mixing Ae. aegypti population of 10,000 adult females and model parameters described in Supplemental Table . Notably, pgSIT releases outcompete those of all other suppression technologies, showing the highest potential to eliminate the local population