## 3 Discussion
### Genetic Engineering and Gene Editing via CRISPR  
The primary purpose of this work was to sequence the _A. domesticus_ genome to obtain necessary sequence data for genetic engineering to improve these animals as a sustainable food resource and for other forms of bioproduction. Thus, we identified and annotated two genes, Ad vermilion and Ad muscle actin, which were used to create knock-out and knock-in strains of _A. domesticus_ as an initial proof of concept to develop the tools required for our downstream technologies. The analysis of Ad vermilion was our first indication that the genome of this species contains quite large introns. The exon sizes were similar to those of other orthologous insect genes such as vermilion from _T. castaneum_, but due to the extended intron length, this gene was more than 100 times longer in _A. domesticus_. The _T. castaneum_ vermilion gene is around 2 kb but is around 90 kb in Ad vermilion. On the other hand, the annotation of the Ad muscle actin gene was more straightforward. As with _Tribolium_ sp. and _Drosophila_ sp., Ad muscle actin has only one exon and, therefore, is similar in size to that of other insect species. Once we had these genes annotated, we were then able to use the Ad vermilion gene to identify CRISPR target sites, and the promoter region from Ad muscle actin gene was harnessed for high level expression of knock-in marker genes. The Ad vermilion gene, when disrupted as a target for knock-in experiments, also functions as a convenient biomarker, as reducing or eliminating function of this gene results in a white-eye (or vermilion color eye) phenotype with less eye pigmentation than wild type. Thus, for the CRISPR genetic engineering work, we showed that the CRISPR/Cas9 gene editing tool works in _A. domesticus_. With up to a 68% CRISPR knock-out rate in G0, the efficiency was sufficient to produce and establish the modified strains for research and future commercial applications. After multiple generations in the laboratory, the vermilion mutation in _A. domesticus_ colonies does not demonstrate any obvious decrease in phenotype or fitness based on survival and growth rate (data not shown). For our knock-in CRISPR experiments, we used the same sgRNA target site as for the knock-outs. As such, we expected the Cas9-sgRNA-created DNA double break in both the insect genome and the knock-in plasmid. Therefore, the EGFP marker gene was likely incorporated into the genome through the non-homologous end joining (NHEJ) DNA repair process. The Ad muscle actin promoter was successfully utilized to express exogenous EGFP in muscle tissue. The G0 somatic knock-in rate was around 2%, and two out of three outcrosses provided EGFP positive G1, indicating the knock-in is efficient in those G0. As this was our first attempt, an optimized protocol will be created in the future to further improve this knock-in rate. Interestingly, the EGFP colonies had different muscle EGFP expression patterns, possibly as a result of a damaged promoter during knock-in since we used NHEJ to create the knock-in or due to epigenetic factors. Based on these results, we successfully showed CRISPR gene editing works to knock-in genes and function in house cricket and creates possibilities for valuable applications going forward.