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10-Minute Presentation

Physiology, Biochemistry, and Toxicology

10-min: PBT, Genetics

Use of CRISPR screening in insect cells to reveal the mechanism of insect toxins

Wednesday, November 13, 2024
4:42 PM – 4:54 PM MST
Location: Phoenix Convention Center, 121 C, PCC
Oral Presentation
Bacterial toxins that selectively kill insects are valuable tools for protecting food crops from insect predation (e.g. Bt toxin). Increasing the toolbox through better mechanistic understanding and engineering could help address off-targeting and resistance, but the mechanisms of many promising insect toxins remain unknown. We previously showed that high resolution genome-wide CRISPR screening in insect cell lines can reveal their mechanisms by finely mapping all possible resistance genes in insect cells (Xu, Viswanatha et al., Nature 2022). We now apply the approach to Nigritoxin, a novel marine Vibrio protein toxin, that kills crustacean and insect cells, posing a threat to shrimp farming and offering potential as a bioinsecticide. To determine the entry and killing mechanism, we created a new platform and performed the first genome-wide CRISPR screen in a mosquito cell line (Anopheles coluzzii) that is highly sensitive to Nigritoxin. The top hit in our screen was a plasma membrane protein which we validated as the Nigritoxin receptor. Structural analysis determined that a β-sheet of the receptor rigidifies and engages a β-strand within the Nigritoxin N-terminal domain. Finally, guided by another hit in the screen, the mosquito ortholog of a ribosome-quality-control (RQC) factor, we found that once inside cells, Nigritoxin inhibits protein translation. Mechanistically, the C-terminal domain of Nigritoxin depurinates a critical adenosine base in the 28S ribosomal RNA potently inhibiting translation in cells and in vitro. In conclusion, CRISPR screening in insect cell lines with custom tailored CRISPR libraries can identity the mechanisms of insect toxin entry and killing.