Genome-Scale Exon Perturbation Screens Uncover Critical Exons for Cell Fitness [CRISPR]
Although CRISPR-Cas technology has revolutionized functional genomics, the systematic exploration of the role of individual exons for critical cellular phenotypes is lagging, limiting our understanding of genome regulation. To overcome this constraint, we have optimized and applied massively parallel exon deletion and splice site mutation screens in human cell lines identifying thousands of exons required for cell fitness. Fitness-promoting exons are enriched in essential and highly expressed genes and frequently overlap protein domains and interaction interfaces. In contrast, fitness-suppressing exons that are enriched in low-expressed, non-essential genes and tend to overlap intrinsically disordered regions. In-depth mechanistic investigation of a screen hit, TAF5 alternative exon-8, reveals that its inclusion controls the assembly of the TFIID general transcription initiation complex regulating gene expression outputs. Collectively, by applying orthogonal exon perturbation screening strategies we have generated a resource of phenotypically important exons and uncovered mechanisms that control gene expression and cell fitness. Overall design: In brief, cells were infected with lentiviral CRISPR libraries, followed by proliferation-basd drop-out screening. Cell pellets were collected at the beginning and end time points, genomic DNA was extracted, hgRNA expression cassettes were amplified and sequencing libraries were prepared with custmorized protocols. The libraries were quantified and sequenced on Illumina sequencing platform.