A natural Nlscarlet insertion underlies the orange-eye phenotype and facilitates efficient multiple-sgRNA genome editing in Nilaparvata lugens.

Abstract

Eye-color mutants provide easily distinguishable visual markers for genetic analysis and genome editing, yet the molecular basis of naturally occurring eye-color variation in Nilaparvata lugens remains unclear. Here, we combined classical genetic analysis, candidate-gene screening, and CRISPR/Cas9 genome editing to elucidate the genetic basis of a stably inherited orange-eye phenotype and evaluate the editing performance of different sgRNA strategies. The orange-eye phenotype was inherited as a single autosomal recessive trait, and the mutant strain showed only limited fitness differences from the WT strain. A 1,287-bp insertion spanning exon 13 and the adjacent intronic region of Nlscarlet introduced a premature stop codon, truncating NlScarlet after the fifth transmembrane helix. The insertion completely cosegregated with the orange-eye phenotype, and CRISPR/Cas9-mediated disruption of Nlscarlet recapitulated the phenotype, demonstrating that this insertion underlies the natural mutation. A separate in-frame deletion of lysine 535 produced a dark-orange-eye phenotype, and structural modeling suggested that this residue may contribute to the interaction between the NlWhite/NlScarlet transporter complex and 3-hydroxykynurenine. Using Nlscarlet as a visually scorable marker, two multiple-sgRNA strategies, each using four sgRNAs, increased the proportions of G0 individuals with complete loss-of-function phenotypes from 30.97% to 84.73% and 92.47%, respectively, and of mutant-eyed G1 progeny from 37.33% to 81.33% and 90.67%, respectively. Heritable mutations generated by both strategies were predominantly large deletions. These findings identify the genetic basis of a natural orange-eye mutation in N. lugens and establish multiple-sgRNA editing as an efficient strategy for rapid functional screening in insects.