## 2 Results
For knock-in experiments, sgRNA#1 and the knock-in construct containing the enhanced green fluorescent protein (EGFP) marker gene coding region (Figure 7) were co-injected with Cas9 protein into young _A. domesticus_ eggs. We injected 939 eggs, and 255 crickets successfully hatched (hatch rate of 27%). From all hatched crickets, we obtained six G0 crickets showing EGFP expression (2%) (Figure 8). The knock-in positive phenotype selected was based on EGFP expression in muscle tissue due to use of the Ad muscle actin promoter. EGFP expression varied from a small group of muscles to more than half of the muscle in the body (Figure 8D,E). The enlarged picture shows a \begin{table}
\begin{tabular}{l c c c c c c c c c c c} \hline \hline \multicolumn{2}{c}{Strain} & \multicolumn{4}{c}{Sequence} & \multicolumn{4}{c}{Deletion (bp)} \\ \hline AA seq & G & G & A & G & R & S & G & R & R & G & A & G & R & \\ \hline Wild Type & & & & & & & & & & & & \\ \hline V2-1 & & & & & & & & & & & & \\ \hline V2-3 & & & & & & & & & & & & \\ \hline V2-6 & & & & & & & & & & & & \\ \hline V2-7 & & & & & & & & & & & & \\ \hline V2-7 & & & & & & & & & & & & \\ \hline V2-7-2 & & & & & & & & & & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Deletions from CRISPR knock-out strains in _A. domesticus_. Figure 6: Vermilion eye color phenotype in _A. domesticus_. (**A**) wild-type eye color in freshly hatched cricket (left) and few hours old cricket (right). (**B**) Eye color of completely knocked-out Ad vermilion gene (left) and cricket with partial knock-out phenotype (right). The white arrows point out the location of the cricket eyes where the respective phenotypes can be observed. more detailed view of the muscle structure with GFP expression in part of the cricket leg (Figure 8F). These results indicate that the promoter is functional and successfully expressing the marker gene in the appropriate tissue. From the G\({}_{0}\) EGFP positives, we were able to set up three out-crosses (with wild-type crickets) and one G\({}_{0}\) EGFP negative cross (using injected hatchlings that did not show EGFP phenotype) to screen G\({}_{1}\) offspring. From these experiments, we received EGFP positive G\({}_{1}\) from two of the positive G\(0\) out-crosses (#1 and #3) and were able to establish two muscle EGFP expression colonies (Figure 9). In Figure 9A,B, we can see that the EGFP expression patterns from #1 and #3 colonies are different. Crickets from colony #1 showed much lower EGFP expression and were only easy to screen from hind leg muscle, but most muscles in the body of crickets from colony #3 were expressing EGFP. There were no EGFP positive G\({}_{1}\) offspring from the negative G\(0\) crosses. Figure 8: Somatic knock-in EGFP in _A. domesticus_ by CRISPR/Cas9. Wild-type cricket under white light (**A**) and florescent light with GFP filter (**B**). Successful knock-in cricket with large area of somatic knock-in under white light (**C**) and florescent light with GFP filter (**D**). Cricket with small area GFP knock-in phenotype, (**E**) and enlarged picture of muscle structure GFP expression (**F**). Figure 7: CRISPR knock-in construct in _A. domesticus_. Ad muscle actin was used to identify the promoter driving EGFP as the marker gene. Two short sequences contain the Ad vermilion sgRNA sites flanking both side of the marker gene. RNAi experiments. In addition to demonstrating CRISPR-Cas9 knock-out and knock-in efficacy in _A. domesticus_ based on our genomic data, we also demonstrated efficacy of RNAi in this species using the same data. Two different target genes were used: (1) Ad vermilion gene (in wild type crickets) and (2) the EGFP gene from CRISPR-Cas9 stable EGFP expressing lines. For each of the two strains, we injected three sets of crickets with each dsRNA (Table 5). The results had survival rates four weeks post injection of approximately 70% for RNAi constructs and 51% for controls.