Genome-wide CRISPR screening in insects to reveal novel regulators of developmental signaling pathways

Developmental patterning in insects is orchestrated by conserved signaling cascades, e.g. the Wnt/Wingless (Wg) pathway, yet comprehensive identification of pathway regulators in arthropods remains incomplete. Fluorescence-activated cell sorting (FACS)-based CRISPR screens offer a new approach to systematically map positive and negative regulators of signaling in insect cells. Here we make a dramatic improvement to the CRISPR screening protocol in Drosophila S2 cells and leverage the optimized approach to perform FACS-based CRISPR screens to identify regulators of Wg signaling. To improve CRISPR screening in insect cells, we devised the Integrase and anti-CRISPR (IntAC) method where we transiently suppress Cas9 activity during sgRNA library integration by transiently transfecting anti-CRISPR whilst stably transfecting Cas9 and sgRNAs. We next explored whether this approach can detect genes whose deletion results in signaling pathway up- or down-regulation. To accomplish this, we stably transfected pathway reporters along with CRISPR sgRNAs. After pathway stimulation, high and low responding populations were collected by FACS and their sgRNAs sequenced. For the Wg pathways, our approach recovered canonical pathway components, e.g. armadillo and dishevelled, and revealed novel enhancers and suppressors. Our findings demonstrate that FACS-based CRISPR screens can be conducted in insect cells, enabling discovery of both enhancers and suppressors of developmental signaling pathways. Importantly, this versatile platform could function in principle in any transfectable cell line, providing a method for genome-wide interrogation of signaling pathways in non-model insects and arthropods.