Fig. 1.

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. ⁶⁸). 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 *)