Genome-Scale Exon Perturbation Screens Uncover Exons Critical for Cell Fitness

CRISPR-Cas technology has transformed functional genomics, yet understanding 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 non-essential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the TAF5 alternative exon-8, screen hit, 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.