Pigmentation markers enable efficient co-CRISPR selection in the pantry moth Plodia interpunctella

CRISPR co-targeting (co-CRISPR) of pigment genes facilitates the identification and recovery of genome-edited individuals in model organisms. Using Plodia interpunctella, an emerging lepidopteran model, we characterized mutant phenotypes of eight genes involved in the melanin and ommochrome biosynthesis pathways (black, ebony, yellow, tan, vermilion, kynurenine 3-monooxygenase/cinnabar (kmo/cn), scarlet, and white) to evaluate their suitability as co-CRISPR markers. Disruption of black, ebony, and yellow altered larval sclerite, pupal cuticle, and adult wing coloration. All four ommochrome pathway genes studied contributed to larval cuticle and adult eye pigmentation, whereas only vermilion and kmo/cn disruption produced visible phenotypes in the adult wings. Sequencing revealed low apparent mosaicism in some yellow and tan G0 individuals, along with recurrent deletions at the yellow and tan target sites consistent with possible microhomology-mediated end joining repair. Using white or scarlet as co-CRISPR selection markers, we identified G0 individuals carrying a secondary mutation in the yellow gene with a positive predictive value of 100% by scoring its knockout phenotype. Extending this approach to six meiosis-related genes without visibly scorable phenotypes, selection based on the white mutant phenotype identified sequence-confirmed mutations at the corresponding gene-of-interest in 191 of 206 G0 individuals (93%). Together, these findings establish white and scarlet as effective co-CRISPR selection markers and provide a practical strategy for facilitating functional genetic studies in Plodia.