Genome-scale exon perturbation screens uncover exons critical for cell fitness

  • Mol Cell. 2024 Jul 11;84(13):2553-2572.e19. doi: 10.1016/j.molcel.2024.05.024.
Mei-Sheng Xiao  1 ,  Arun Prasath Damodaran  2 ,  Bandana Kumari  1 ,  Ethan Dickson  1 ,  Kun Xing  1 ,  Tyler A On  3 ,  Nikhil Parab  1 ,  Helen E King  4 ,  Alexendar R Perez  5 ,  Wilfried M Guiblet  1 ,  Gerard Duncan  6 ,  Anney Che  7 ,  Raj Chari  8 ,  Thorkell Andresson  6 ,  Joana A Vidigal  9 ,  Robert J Weatheritt  10 ,  Michael Aregger  11 ,  Thomas Gonatopoulos-Pournatzis  12
Affiliations
  • 1. RNA Biology Laboratory, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD 21702, USA.
  • 2. RNA Biology Laboratory, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD 21702, USA. Electronic address: [email protected].
  • 3. Molecular Targets Program, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD 21702, USA.
  • 4. EMBL Australia and Garvan Institute of Medical Research, Sydney, NSW 2010, Australia.
  • 5. Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD 20892, USA; Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, CA 94143, USA.
  • 6. Protein Characterization Laboratory, Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD 21701, USA.
  • 7. Advanced Biomedical Computational Science, Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD 21701, USA.
  • 8. Genome Modification Core, Frederick National Laboratory for Cancer Research (FNLCR), Frederick, MD 21702, USA.
  • 9. Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD 20892, USA.
  • 10. EMBL Australia and Garvan Institute of Medical Research, Sydney, NSW 2010, Australia; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2010, Australia.
  • 11. Molecular Targets Program, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD 21702, USA. Electronic address: [email protected].
  • 12. RNA Biology Laboratory, Center for Cancer Research (CCR), National Cancer Institute (NCI), National Institutes of Health (NIH), Frederick, MD 21702, USA. Electronic address: [email protected].
PMID: 38917794 DOI: 10.1016/j.molcel.2024.05.024
Abstract

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.

Keywords
CRISPR screen; Cas12a; TAF5; TFIID; alternative splicing; base editor; cell fitness exons; exon deletion; exon perturbation; functional genomics.
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