Genome editing is a critical tool for studying functional genomics to elucidate the biochemical mechanisms and catabolic pathways necessary for metabolizing toxins or insecticides in pest insects. The advent of the Clustered Regularly Interspaced Short Palindromic Repeats/(CRISPR)-associated protein 9 (CRISPR/Cas9) system provides a widely applicable novel genome-editing tool with advantages over techniques such as zinc finger nucleases and transcription activator-like effector nucleases that are typically more time consuming and labor intensive (Miller et al., 2007, Miller et al., 2011, Porteus and Baltimore, 2003, Sander et al., 2011, Wood et al., 2011). The CRISPR/Cas9 system targets and cleaves a specific DNA sequence using the Cas9 nuclease coupled with a single guide RNA (sgRNA) (Sander et al., 2011). The site-specific double‐stranded DNA break is then repaired either by non‐homologous end joining (NHEJ) or by homology-directed recombination (HDR) in the presence of a donor DNA template. The CRISPR/Cas9 system has been used to precisely edit genomes of numerous eukaryotic organisms (Cho et al., 2013, Cong et al., 2013, Ding et al., 2013, Hwang et al., 2013, Ran et al., 2013, Wood et al., 2011) including the insect orders Blattodea (Shirai et al., 2022), Coleoptera (Gilles et al., 2015), Diptera (Bassett and Liu, 2014, Bassett et al., 2013, Hall et al., 2015, Kistler et al., 2015), Hemiptera (Cagliari et al., 2020), Homoptera (Heu et al., 2020), Hymenoptera (Chaverra‐Rodriguez et al., 2020; Dalla Benetta et al., 2020, Nie et al., 2021), Lepidoptera (Li et al., 2015, Wang et al., 2013, Wei et al., 2014), and Orthoptera (Li et al., 2016). The CRISPR biotechnology has facilitated functional genomic studies of model and non-model species and the development of novel genetics-based pest control approaches (for reviews, see Taning et al., 2017, Li et al., 2021, Suresh et al., 2021).

To validate CRISPR/Cas9-mediated gene editing in the Indian meal moth, Plodia interpunctella Hübner (Lepidoptera: Pyralidae), the white gene was selected as the initial target. The white, brown and scarlet genes encode for ABC transporter proteins that are involved in the movement of ommochrome and pteridine pathway precursors into pigment granules in the eye (Bretschneider et al., 2016, Denecke et al., 2017, Ewart and Howells, 1998, Heckel, 2012, Khan et al., 2017). Dimerization of White and Scarlet proteins in Drosophila melanogaster (Mackenzie et al., 1999) and the silk moth, Bombyx mori (Osanai-Futahashi et al., 2016, Quan et al., 2002, Tatematsu, 2011), leads to the transport of the ommochrome precursors while dimerization of White with Brown protein affects the transport of the pteridine precursors. These and other studies in Lepidoptera showed that mutations within the white locus are typically non-lethal (Li et al., 2015, Wang et al., 2013, Wei et al., 2014). However, this is not the case for all Lepidoptera especially the Noctuidae where gene editing of the white locus in Helicoverpa armigera with CRISPR/Cas9 led to an embryonic lethal phenotype (Khan et al., 2017). This is in contrast with gene-edited mutations of the brown or scarlet loci which produced viable offspring with the respective eye color phenotypes in H. armigera.

Assessing the white locus in P. interpunctella may help elucidate the mechanisms of resistance to Bacillus thuringiensis (Bt) toxin in this insect. P. interpunctella is a major economic pest of stored products and processed foods worldwide (Mohandass et al., 2007). Control of this pest has become more difficult with the increasing prevalence of resistance to most chemical pesticides in a number of populations. Resistance has also been observed against biopesticides in bioengineered crops. The first observation of acquired B. thuringiensis (Bt) resistance was made after laboratory selections involving continuous feeding of P. interpunctella on Bt treated food led to a 100-fold decrease in susceptibility within fifteen generations (McGaughey, 1985). In Plutella xylostella, a naturally occurring mutant of a novel white ortholog, Pxwhite, resulted in transcript down regulation that caused reduced Cry1A toxin susceptibility (Guo et al., 2015). Furthermore, RNA interference (RNAi)-mediated suppression of the White gene in P. xylostella resulted in significantly reduced larval susceptibility to Cry1Ac (Guo et al., 2015).

A non-lethal, recessive autosomal white strain has been isolated in P. interpunctella (Piw-) and found to be the result of a single nucleotide deletion at c.737delC that resulted in a frameshift mutation (Heryanto et al., 2022). Previously, CRISPR/Cas9 was used to produce somatic mutations to Ultrabithorax in Junonia coenia, Vanessa cardui and P. interpunctella in order to assess the homeotic phenotypes produced in wings (Tendolkar et al., 2021) and to demonstrate CRISPR HDR repair of the c.737delC mutation (Heryanto et al., 2022). To assess the functionality of white in P. interpunctella and to determine the possibility of establishing germline mutations, CRISPR/Cas9 gene editing was conducted in this study. Heritable mutations of the white gene were isolated and characterized demonstrating the utility of gene-editing in this moth.