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

Abstract

The western tarnished plant bug, Lygus hesperus, is a key hemipteran pest of numerous agricultural, horticultural, and industrial crops in the western United States and Mexico. A lack of genetic tools in L. hesperus hinders progress in functional genomics and in developing innovative pest control methods such as gene drive. Here, using RNA interference (RNAi) against cardinal (LhCd), cinnabar (LhCn), and white (LhW), we showed that knockdown of LhW was lethal to developing embryos, while knockdown of LhCd or LhCn produced bright red eye phenotypes, in contrast to wild-type brown eyes. We further used CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) genome editing to generate germline knockouts of both LhCd (Card) and LhCn (Cinn), producing separate strains of L. hesperus characterized by mutant eye phenotypes. Although the cardinal knockout strain Card exhibited a gradual darkening of the eyes to brown typical of the wild-type line later in nymphal development, we observed bright red eyes throughout all life stages in the cinnabar knockout strain Cinn, making it a viable marker for tracking gene editing in L. hesperus. These results provide evidence that CRISPR/Cas9 gene editing functions in L. hesperus and that eye pigmentation genes are useful for tracking the successful genetic manipulation of this insect.

CRISPR/Cas9-mediated genome editing ofFrankliniella occidentalis,the western flower thrips, via embryonic microinjection

“…Unlike the Fo-w knockout line, which displays an age-dependent knockout phenotype, the Fo-cn knockout line exhibited the same phenotype throughout all life stages, that of bright red eyes ( Figure 1 ). Similar knockout phenotypes have been reported for cn in other insect systems (Heu et al, 2022; Sullivan et al, 1979; Vargas-Lowman et al, 2019; Xue et al, 2018). For example, while the brown planthopper eye usually appears brown, mutagenesis of the Nl - cinnabar gene led to a striking transformation to bright red (Xue et al, 2018).…”

Genome editing of thevermilionlocus generates a visible eye color marker forOncopeltus fasciatus

“…Knockdown of Lh-bw suggested that the pteridine pathway contributes to eye color while w knock down impacted both eyes and body, with phenotypes including smaller and paler bodies and soft cuticles. Subsequent CRISPR/Cas9 mutagenesis in this species showed that the ommochrome pathway components Lhcn and Lh-cd are both involved in eye pigmentation, with Lh-cn mutants displaying sex-specific phenotypic differences in adults 38 . In the brown planthopper, Nilaparvata lugens, CRISPR/Cas9 genome editing suggested that Nl-w and ommochrome pathway member Nl-cn are necessary for wild type eye color 39 .…”

“…In our first attempt to generate a visible eye color marker for O. fasciatus , we chose to target Of - w , given its historic use as a visible marker 49 . While that approach established CRISPR/Cas9 mutagenesis for O. fasciatus , we were surprised to find lethality in animals homozygous for white null alleles 9 , although w had been shown to be required for viability in the moth Helicoverpa armigera 50 and more recently in L. hesperus 38 . Here, we used RNAi to assess roles of selected genes encoding candidate visible markers, testing for detrimental consequences of gene knockdown before proceeding to genome editing.…”

First report on the utility of pupal case for early determination of CRISPR/Cas9 ribonucleoprotein mediated genomic edits in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera)

“…Unlike in plants, one cannot use invasive method for edit characterization as individual flies carry unique edits due to the random nature of the double‐stranded DNA break repair mechanism and also limited by the low success rate of microinjection (Chaverra‐Rodriguez et al, 2020; Heu et al, 2022; Koidou et al, 2020). In addition to the above, maiming test animals are not permitted to determine the type of genomic edits in the treated individuals.…”

“…Rendering sequencing is the ultimate method to determine the nature of the edit/s (Bi et al, 2022; Fu et al, 2022; Zhang et al, 2015). In this regard, it is relatively easy to identify the genomic edits in phenotypic genes, such as white , that play a vital role in eye color pigmentation, where the edited flies bear white eyes as compared to the wild eye color (Bai et al, 2019; Chaverra‐Rodriguez et al, 2020; Choo et al, 2018; Khan et al, 2017; Koidou et al, 2020, Heu et al, 2022). But the same is not possible for the non‐phenotypic, functional genes mentioned above, requiring 2–3 months of wait time to know whether or not editing has happened.…”