Professor & Department Chair University of Maryland College Park, Maryland
Visible markers facilitate genetic analysis of model organisms. We will discuss our efforts to develop lines with visible mutations in the large milkweed bug Oncopeltus fasciatus, a re-emerging model hemipteran species. We developed CRISPR/Cas9 germline editing to generate visible markers and mutations in other genes of interest in Oncopeltus. In our first study, we found that the white gene, which serves as a marker in phylogenetically diverse insect species, is not a useful marker for Oncopeltus because mutations result in lethality for homozygotes (Reding et al. 2020). We next used RNAi to test the function of several other pigmentation genes in this species to identify a gene whose loss results in a clear visible phenotype without detriment to viability. We found that Of-vermilion (Of-v), which yields red eyes when knocked down, fits these criteria. We used CRISPR/Cas9 to generate mutations at the v locus and established three independent Of-v red-eyed lines that are viable and fertile (Reding et al. 2023). We have used these Of-v lines to perform CRISPR/Cas9 co-mutation to isolate loss-of-function alleles for a novel segmentation gene. All mutant alleles were isolated from G0s that also gave rise to Of-v progeny, suggesting that co-mutation is an efficient strategy when screening for mutant alleles that do not cause a visible phenotype, particularly when Cas9 activity is not germline-restricted. While development of these and other genetic tools for Oncopeltus has expanded the utility of this non-holometabolous insect as a model system, long-term maintenance of homozygous-lethal mutations remains a challenge.
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