Single-Cell Exon Deletion Profiling Reveals Splicing Events That Shape Gene Expression and Cell State Dynamics

Published: 9 January 2026| Version 1 | DOI: 10.17632/rxfzrptr2d.1
Contributors:
,
,
, Mei-Sheng Xiao, Amit K. Behera, Tyler A. On, Carl E. McIntosh, Maxwell Teszler, Chelsee Holloway, Sandra Le, Nikhil Parab, Yongmei Zhao, Michael Aregger, Thomas Gonatopoulos Pournatzis

Description

Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we developed scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identified numerous exons with pronounced regulatory effects on gene expression and cell cycle progression. Detailed analysis of the alternative NRF1 exon-7 demonstrated that its inclusion modulates NRF1’s regulatory function by influencing its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a robust and versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.

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Institutions

  • National Cancer Institute
  • National Institutes of Health
  • Center for Cancer Research

Categories

Confocal Microscopy, Flow Cytometry, Western Blot, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis

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