Genome-Scale Exon Perturbation Screens Uncover Critical Exons for Cell Fitness [RNA-seq]

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression. Overall design: RNA-Sequencing was performed in HEK293 Flp-in cells depleted from endogenous TAF5 with RNAi (non-targeting siRNAs were used as control) while expressing either full-length TAF5 (TAF5-FL) orthe TAF5 isoform lacking exon-8 (TAF5-?E8)at endogenous levels using dox-inducible HEK293 Flp-In cell lines to systematically explore if TAF5 exon-8 regulates gene expression.