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

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. The GUIDE-seq experiment was conducted according to a well-established protocol with minor adjustments, as detailed in a relevant publication (PMID: 34773119). Double-stranded dsODN was synthesized by annealing two modified oligonucleotides (PMID: 30654827). RPE1 cells, engineered to stably express both Cas9 and Cas12a nucleases, were transfected with dsODN and subsequently infected with viruses carrying various hgRNAs. Following puromycin selection, genomic DNA (gDNA) was isolated, sheared, and subjected to library preparation for high-throughput sequencing using NextSeq 1000/2000.