Affiliation: Division of Biological Sciences, Section of Cell and Developmental Biology, UC San Diego, La Jolla, California, 92092 USA
Keywords: g. CRISPR/Cas9; e. gene targeting and modification
Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females before male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile, fit, and competitive males, we previously described a CRISPR-mediated precision-guided Sterile Insect Technique (pgSIT). PgSIT functions by exploiting the precision and accuracy of CRISPR to simultaneously disrupt genes essential for female viability and male fertility. It utilizes a simple breeding scheme requiring two strains: one expressing Cas9 and the other expressing multiple guide RNAs (gRNAs). A single mating between these strains mechanistically results in synchronous Cas9/RNA-mediated dominant biallelic knockouts of target genes throughout development, resulting in the complete penetrance of desired phenotypes in all progeny. This two-locus pgSIT design was used to engineer pgSIT systems in Drosophilamelanogaster, Drosophila suzukii, a global pest of economically important soft-skinned fruits, and Aedes aegypti, the principal vector for arboviruses including dengue/yellow fever, chikungunya, and Zika virus. While effective at generating F1 sterile males, the two-locus pgSIT depends on a genetic cross between the two parental strains which requires maintenance and sexing of two strains in a factory. Therefore, to further advance pgSIT by removing this crossing step, we describe a one-locus Temperature-Inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in D.melanogaster. Importantly, we were able to develop a true-breeding strain for TI-pgSIT that eliminates the requirement for sex sorting, a feature that may help further automate production at scale.