Metabolic pathway interruption: CRISPR/Cas9-mediated knockout of tryptophan 2,3-oxygenase in Tribolium castaneum

Authors: Adrianos, Sherry; Oppert, Brenda; LORENZEN, MARCE - North Carolina State University

Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/14/2018
Publication Date: 3/15/2018
Citation: Adrianos, S.L., Oppert, B.S., Lorenzen, M. 2018. Metabolic pathway interruption: CRISPR/Cas9-mediated knockout of tryptophan 2,3-oxygenase in Tribolium castaneum. Journal of Insect Physiology. 107:104-109. https://doi.org/10.1016/j.jinsphys.2018.03.004.
DOI: https://doi.org/10.1016/j.jinsphys.2018.03.004

Interpretive Summary: The red flour beetle is a major pest of grain and grain based foods such as flour, but has also emerged as a sophisticated model system for studying evolutionary biology and development. This genetic research can also lead to new biologically-based insecticides to control these pests. One of the first steps in bringing transgenic technologies to new species is the development of efficient markers to indicate when transformation has occurred, and changes in eye color can be useful markers. Red flour beetles normally have black eyes, but we used a new technology called CRISPR to block eye coloring pigment production in the beetles and generate eye-color mutants. Without the black coloring in the eyes, the eyes are now white. CRISPR utilizes the cell’s own repair machinery to repair a precise cut made by a nuclease directed by a guide RNA, and our research also showed that guides differed in their efficiency. Molecular analysis by target site sequencing and PCR diagnostic assays illustrated that the nuclease was able to generate small, targeted deletions in the eye color gene. These white-eyed beetles are being used in genetic studies to develop new biologically-based products to reduce the damage caused by these and other stored product beetle pests. This technique can also be used to create white eyed mutants in other insect species.

Technical Abstract: The Tribolium castaneum vermilion gene encodes tryptophan 2,3-dioxygenase, a pivotal enzyme in the ommochrome pathway that is responsible for the black eye color. T. castaneum strains with a loss-of-function mutation, vermilion white (vw), lack both the promoter and the first 80% of the vermilion coding sequence, resulting in an absence of eye pigmentation and a white-eyed phenotype. In this report, the CRISPR/Cas9-nuclease system had been used to deliver a guide RNA targeting vermilion in early stage T. castaneum embryos, resulting in mutations in the gene. As adults, injected beetles have black eyes; however, after mating with vw, 19 percent of the progeny from one injected individual had white eyes. Molecular analysis by target site sequencing and PCR diagnostic assays illustrated that the nuclease was able to generate small, targeted deletions in vermilion. Black-eyed progeny had a six bp deletion, with presumably deletion of two amino acids and functional vermilion. White-eyed individuals had a two bp deletion that resulted in a frameshift mutation and nonfunctional vermilion. T. castaneum progeny with effective CRISPR vermilion knockout were self-mated to isolate and establish a mutant CRISPR-generated line of beetles with a white-eyed phenotype for future work. The data indicate that the CRISPR/Cas9 system can be used to generate loss-of-function vermilion alleles to generate white-eyed strains in other species for downstream applications.