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10-Minute Presentation

Physiology, Biochemistry, and Toxicology

10-min: PBT, Genetics

CRISPR knockout screening in insect and tick cells

Wednesday, November 13, 2024
1:42 PM – 1:54 PM MST
Location: Phoenix Convention Center, 121 C, PCC
Oral Presentation
CRISPR-Cas9 knockout screening in cultured cells provides a powerful and efficient approach to gene function discovery in model and non-model species. We previously established CRISPR-Cas9 knockout screening in Drosophila melanogaster cells and used the platform to uncover new findings, including identification of a transporter of ecdysone and receptors of bacterial toxins. Here, we present our successful efforts to extend the platform to non-model species, opening the doors to unbiased genetic screening in cells from vectors of human diseases and other arthropods. We have fully established CRISPR knockout screening in Anopheles coluzzii mosquito cells and performed genome-wide screens to detect genes required for growth or viability, as well as more specialized cellular functions. We have also made progress establishing the platform for cells from Ixodes scapularis, a tick vector of Lyme and other human diseases. As part of these efforts, we developed bioinformatics resources in the form of genome-wide collections of sgRNA designs for Anopheles coluzzii, Anopheles gambiae, Anopheles stephensi, Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus and for I. scapularis. We further used our DIOPT integrated multi-algorithm approach to map orthologs among D. melanogaster, mosquitos, lepidopteran pest species, other insects, and I. scapularis. Altogether, we have engineered arthropod cells, developed reagent libraries, and built bioinformatics resources that facilitate CRISPR screens and other studies. The CRISPR screen approach will be useful to identify receptors, transporters, and mechanism of action of biotoxins or insecticides; genes required for host-pathogen interactions; and, more broadly, genes required for diverse cellular activities.