Decoding post-transcriptional regulatory networks by RNA-linked CRISPR screening in human cells

  • Nat Methods. 2025 Jun;22(6):1237-1246. doi: 10.1038/s41592-025-02702-6.
Patrick J Nugent  1  2 ,  Heungwon Park  1 ,  Cynthia L Wladyka  3 ,  James N Yelland  1 ,  Sayantani Sinha  4 ,  Katharine Y Chen  1  2 ,  Christine Bynum  1  5 ,  Grace Quarterman  1  5 ,  Stanley C Lee  4 ,  Andrew C Hsieh  3  6 ,  Arvind Rasi Subramaniam  7  8
Affiliations
  • 1. Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA, USA.
  • 2. Molecular and Cellular Biology Graduate Program, University of Washington, Seattle, WA, USA.
  • 3. Human Biology Division, Fred Hutchinson Cancer Center, Seattle, WA, USA.
  • 4. Translational Science and Therapeutics Division, Fred Hutchinson Cancer Center, Seattle, WA, USA.
  • 5. Department of Biology, Spelman College, Atlanta, GA, USA.
  • 6. Department of Medicine and Department of Genome Sciences, University of Washington, Seattle, WA, USA.
  • 7. Basic Sciences Division and Computational Biology Section of the Public Health Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA, USA. [email protected].
  • 8. Department of Biochemistry and Department of Genome Sciences, University of Washington, Seattle, WA, USA. [email protected].
PMID: 40442371 DOI: 10.1038/s41592-025-02702-6
Abstract

RNAs undergo a complex choreography of metabolic processes that are regulated by thousands of RNA-associated proteins. Here we introduce ReLiC, a scalable and high-throughput RNA-linked CRISPR approach to measure the responses of diverse RNA metabolic processes to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC relies on an iterative strategy to integrate genes encoding Cas9, single-guide RNAs (sgRNAs) and barcoded reporter libraries into a defined genomic locus. Combining ReLiC with polysome fractionation reveals key regulators of ribosome occupancy, uncovering links between translation and proteostasis. Isoform-specific ReLiC captures differential regulation of intron retention and exon skipping by SF3B complex subunits. Chemogenomic ReLiC screens decipher translational regulators upstream of messenger RNA (mRNA) decay and identify a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a powerful framework for discovering and dissecting post-transcriptional regulatory networks in human cells.

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