Whole-genome RNAi screen to identify modulators of prometastatic gene ASAP1

Authors

• S Claffey
• S Lin
• Y Chen
• PA Randazzo
• K Cheng

Abstract

High expression of the Arf GTPase activating protein ASAP1 correlates with metastasis and poor prognosis in several cancers, including uveal melanoma and soft tissue sarcomas. ASAP1 is a known regulator of actin structures and mesenchymal tissue differentiation. The pathways that regulate ASAP1 expression are largely unexplored despite its correlation with poor prognosis in cancers with high expression levels. To identify modulators of ASAP1, we performed a whole genome RNAi screen using a ASAP1-HiBiT CRISPR knock-in reporter U2OS cell line to find genes when inhibited by siRNA would repress the expression of ASAP1 with minimal effects on cell viability. HiBiT is a small 11 amino acid, epitope tag capable of producing a robust bioluminescent signal when bound to its complementation partner, LgBiT. Thus, HiBiT knock-in technology enables quantitative analysis of endogenous protein expression. After optimizing the screening conditions, we first conducted a pilot kinome RNAi screen to evaluate the quality of the assay in a 384-well high-throughput format, and then a whole-genome RNAi screen targeting 21,584 human genes was executed. The average Z’ between non-target and killer siRNAs was 0.67, indicating a robust assay with excellent transfection efficiency. The top gene candidate identified from the screen was ASAP1, again demonstrating the quality of the screen. The resulting gene hits from this screen will provide insight into ASAP1 regulation and are potential therapeutic targets.

Scientific Focus Area: Cancer Biology

This page was last updated on Tuesday, August 6, 2024