CRISPR Tiling

PROJECT SUMMARY- CORE 2 Fusion oncoproteins (FOs) enclose folded domains and intrinsically disordered regions (IDRs) rearranged from fusion partner genes, contributing to condensate formation and oncogenic transformation. Understanding of functional elements within FOs will elaborate on how FOs promote aberrant gene expression and other types of chromatin deregulation in cellular cancer model systems. The objective of Core 2 is to serve as a genetic platform for evaluating the FOs under study in Projects 1, 2, and 3. Based on the recently reported “CRISPR/Cas9 screening of protein domains”, Core 2 has optimized a high-density CRISPR gene scan pipeline termed “CRISPR tiling.” This platform uses libraries of sgRNAs to exhaustively and individually disrupt all 2-3 amino acid long regions within proteins of interest, followed by testing for the contribution of the disrupted region to protein function. The proposed CRISPR tiling strategy is innovative because it will provide a comprehensive and non- biased evaluation of FO coding regions, thereby pinpointing critical elements within folded domains and IDRs for FO-driven malignancies. The impact of Core 2 will be significant as it will assist with Projects 1, 2, and 3 in identifying novel mechanisms and therapeutic opportunities within the investigated FOs.