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<p>Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we develop 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 identify numerous exons with pronounced regulatory effects on gene expression and cell cycle progression. Analysis of the alternative NRF1 exon-7 demonstrates 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 versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.</p>

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Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年2月3日

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  • Bandana Kumari,
  • Arun Prasath Damodaran,
  • Wilfried M. Guiblet,
  • 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

摘要

Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we develop 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 identify numerous exons with pronounced regulatory effects on gene expression and cell cycle progression. Analysis of the alternative NRF1 exon-7 demonstrates 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 versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.