High-Efficiency CRISPR/Cas9 Mutagenesis of the Gene in the Milkweed Bug s.

In this manuscript, we report that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is highly efficient in the hemipteran The gene is well characterized in where mutation causes loss of eye pigmentation; is a reliable marker for transgenesis and other genetic manipulations. Accordingly, has been targeted in a number of nonmodel insects to establish tools for genetic studies. Here, we generated mutations in the () locus using CRISPR/Cas9. We found that is required for pigmentation throughout the body of , not just the ommatidia. High rates of somatic mosaicism were observed in the injected generation, reflecting biallelic mutations, and a high rate of germline mutation was evidenced by the large proportion of heterozygous G1s. However, mutations are homozygous lethal; G2 homozygotes lacked pigment dispersion throughout the body and did not hatch, precluding the establishment of a stable mutant line. Embryonic and parental RNA interference (RNAi) were subsequently performed to rule out off-target mutations producing the observed phenotype and to evaluate the efficacy of RNAi in ablating gene function compared to a loss-of-function mutation. RNAi knockdowns phenocopied homozygotes, with an unusual accumulation of orange granules observed in unhatched embryos. This is, to our knowledge, the first CRISPR/Cas9-targeted mutation generated in While we were unable to establish as a useful visible marker for , these findings are instructive for the selection of visible markers in nonmodel species and reveal an unusual role for an ortholog of a classic gene.