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

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Alternative splicing diversifies the human proteome, playing a key role in gene regulation.
• Understanding the functional impact of alternative splicing on cell fate is crucial.

Purpose of the Study:

• To develop a scalable CRISPR-based screening platform for investigating alternative splicing.
• To systematically link specific alternative splicing events to cellular phenotypes.

Main Methods:

• Developed scCHyMErA-Seq, integrating CRISPR-based exon deletion with 10x Genomics single-cell transcriptomics.
• Enabled simultaneous capture of guide RNAs and polyadenylated transcripts at single-cell resolution.
• Applied the platform for high-throughput profiling of alternative cassette exons.

Main Results:

• Identified numerous exons significantly impacting gene expression and cell cycle progression.
• Demonstrated that NRF1 exon-7 inclusion modulates NRF1's recruitment to target gene promoters.
• Validated scCHyMErA-Seq findings against traditional orthogonal methods, showing enhanced scalability and efficiency.

Conclusions:

• scCHyMErA-Seq is a versatile platform for dissecting the functional consequences of alternative splicing.
• Directly links specific splicing variants to transcriptional phenotypes.
• Facilitates systematic unraveling of alternative splicing's role in cell fate determination.