Genome-Scale Exon Perturbation Screens Uncover Exons Critical for Cell Fitness
Description
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.
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Cells were plated on LAB-TEKII chamber slides (Life Technologies #154534PK) and fixed with 4% paraformaldehyde (ThermoFisher Scientific #28908) for 15 minutes at room temperature. Cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich #T8787) for 10 minutes and blocked with 5% BSA (Sigma-Aldrich #A9647) for 1 hour. Cells were incubated with primary antibodies: SpCas9 (1:500, Diagenode #C15310258) and Myc tag (1:400, Sigma-Aldrich #M4439) for 1 hour at room temperature in DPBS supplemented with 5% BSA. Following washing, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibodies (1:1,000, ThermoFisher Scientific #A32731) or Alexa Fluor 647-conjugated goat anti-mouse antibodies (1:800, ThermoFisher Scientific #A32728) for 1 hour at room temperature in DPBS supplemented with 5% BSA. Nuclear staining was performed with Hoechst 33342 (1:10,000, ThermoFisher Scientific #62249), and the slides were mounted using Fluoromount-G™ Mounting Medium (ThermoFisher Scientific #00-4958-02). Images were acquired using a 63x oil immersion objective on a Leica TCS SP8 confocal microscope and processed using Fiji (ImageJ) software.