High-Efficiency CRISPR/Cas9 Mutagenesis of the white Gene in the Milkweed Bug Oncopeltus fasciatus

Abstract

Here we show that CRISPR/Cas9 is highly efficient in the hemipteran Oncopeltus fasciatus. We chose to target the Of-white (Of-w) locus to establish methods and with the goal of generating a visible marker for future experiments. The white gene is well-characterized in Drosophila where mutation causes loss of eye pigmentation; white is a reliable marker for transgenesis and other genetic manipulations. Accordingly, white has been targeted in a number of non-model insects to establish tools for genetic studies. We found that Of-w is required for pigmentation throughout the body of Oncopeltus, 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, Of-w 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 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 Of-w 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 Oncopeltus. While we were unable to establish white as a useful visible marker for Oncopeltus, these findings are instructive for selection of visible markers in non-model species and revealed an unusual role for an ortholog of a classic Drosophila gene.

RNAi and CRISPR/Cas9 as Functional Genomics Tools in the Neotropical Stink Bug, Euschistus heros

“…A possible explanation could be the unknown role of yel during embryogenesis in E. heros, where complete knockout of yel is lethal. In a similar study in the large milkweed bug Oncopeltus fasciatus, the knock-out of the white gene resulted in significant embryonic mortality [35], although its homolog in Drosophila melanogaster is widely known to be involved in eye pigmentation [36]. Depending on the stage of embryonic development when CRISPR/Cas9 components are delivered, it is not uncommon to have a mixed population of edited (somatic mutations) and unedited cells that can result to mosaic effects in generation 0 (G 0 ) [37].…”

Neofunctionalization of a second insulin receptor gene in the wing-dimorphic planthopper, Nilaparvata lugens

“…Genomic DNA (gDNA) was isolated from whole BPH body or forewings to determine the heterozygous or homozygous genotypes of the BPHs. gDNA was extracted from one individual adult, as reported previously [ 63 ]. Briefly, one adult was homogenized in a 0.2-ml Eppendorf tube, followed by the addition of 50 μl of extraction buffer (10 mM Tris-HCl pH 8.2, 1 mM EDTA, 25 mM NaCl, 0.2 mg/ml proteinase K).…”

CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug

“…Current knowledge of eye pigmentation in Hemiptera is limited to just a few species and has been produced primarily through genetic manipulation of white , cinnabar , and cardinal 20 , 24 , 28 – 30 . In L. lineolaris , a mutant red eye phenotype occurs naturally under field and laboratory conditions 6 , 31 , 32 , although the genetic basis of this phenotype is unknown.…”