## 2 Results
castaneum_ vermition amino acid sequence [56]. An incomplete mRNA was identified as the Ad vermition mRNA. We then used this mRNA sequence to annotate the Ad vermition gene and map the intron-exon splicing sites to identify each exon. The Ad muscle actin gene and its promoter were identified the same way initially using the _T. castaneum_ muscle actin gene. In addition, we were able to identify two very similar actin genes in the _A. domesticus_ genome. Since muscle actin genes in many insect species are a single exon gene, we identified one likely muscle actin gene with the highest score from blast and having a single exon. Once the gene was identified, we searched the genomic sequence 1.5 kb upstream from the start codon for promoter regions (Figure 5b). We targeted the Ad vermition gene to also serve as a convenient visual marker (white/vermilion eye color phenotype) for preliminary identification of positive knock-out/knock-in G0 crickets. The Ad muscle actin gene's promoter was utilized due to its high level of continuous expression in all life stages, as well as its characteristic muscle anatomy expression phenotype, which is easily identified. For our downstream goals of expressing GOI in farm raised insects for food, feed, and bioproduct manufacturing, it is usually critical to use promoters with high levels of GOI expression (e.g., obtain the most product of interest per kilogram of insect) as well as have visual biomarkers (e.g., white eye or EGFP phenotype maintained through breeding selection) for simple genotype maintenance in farms and laboratories. CRISPR knock-in/out experiments. Three sgRNA sites were identified as targets for our CRISPR knock-in/knock-out constructs (Table S8). The promoter of Ad muscle actin has many predicted promoters (Figure 5b). To keep the knock-in DNA construct size small, we used 455 bp of the putative promoter sequences upstream from the starting code as the promoter for the marker gene. For knock-outs, we targeted Ad vermilion using CRISPR/Cas9. Using this target sequence, we designed and used either (1) a combination of 3 different sgRNA or (2) a single sgRNA (sgRNAA#1) (Table 3). The resulting G0 hatchling crickets that received all three sgRNA had 68% eye color knock-out phenotype while 32% of those receiving only sgRNA#1 had the phenotype. This phenotype resulted in eye color change from black (wild type) to vermilion or white eye color (Figure 6A,B). Our results show the knock-out color appears white in freshly hatched crickets and gradually changes to vermilion color after a few hours. In each set of injection experiments, both partial and complete knock-out of eye color phenotypes were found (Figure 6B). Partial eye color knock-out was mosaic. In comparison, the eye color in wild-type crickets were always black even in freshly hatched individuals (Figure 6A). To generate stable lines, groups of positive G0's were subsequently self-crossed. For self-crosses, all 16 crosses and 9 out of 10 crosses from three  
Figure 5: Design of CRISPR target in _A. domesticus_. (**a**) Schematic of the annotated Ad vermilion gene from _A. domesticus_ genome data. Red box: ATG starting code; Yellow boxes: exons; Purple arrow: sgRNA site. Note: in this figure intron sizes are not proportional to exons; (**b**) Ad muscle actin gene with promoter areas. Red box: Sequences used for promoter prediction; Green sequences: predicted promoters; Red sequences: start codon; Yellow sequences: protein coding area; Gary blue area: promoter sequence used in CRISPR knock-in construct. and one sgRNA treatments, respectively, provided vermilion eye color G1s, indicating the CRISPR/Cas9 knock-out rate was particularly high in _A. domesticus_. To check the knock-out sequences, we tested five of the knock-out strains (G2) from the single sgRNA treatment. The sequencing results indicated deletions ranging from just a few base pairs to more than 240 bp among these five strains (Table 4).