Transcriptome-wide profiling of alternative splicing regulators with CRISPore-seq.

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Alternative splicing generates diverse RNA isoforms, but current single-cell CRISPR screens lack isoform-level resolution.
• Existing methods cannot detect changes in alternative splicing or identify specific transcript isoforms after genetic perturbations.

Purpose of the Study:

• To develop a novel method, CRISPore-seq, for simultaneous genetic perturbation and isoform-level transcriptomics in single cells.
• To overcome the limitations of gene-level quantification in current single-cell screening approaches.

Main Methods:

• CRISPore-seq couples massively-parallel CRISPR perturbations with joint short- and long-read transcriptomics.
• Simultaneously captures genetic perturbations, gene expression, full-length transcripts, and surface proteins in single cells.
• Utilizes long reads to identify transcript isoforms with high resolution.

Main Results:

• CRISPore-seq identifies 80% more transcript isoforms compared to short reads, with most long reads mapping to unique isoforms.
• Thousands of perturbation-driven alternative splicing events (ASEs) were identified upon knocking down 15 RNA-binding proteins (RBPs).
• Loss of SF3B4 triggers CCND1 exon skipping, disrupting cell-cycle progression, which is rescued by a CCND1 isoform with the skipped exon.

Conclusions:

• CRISPore-seq provides isoform-level resolution for understanding the impact of genetic perturbations on the human transcriptome.
• This scalable tool links genes to transcriptional phenotypes, enabling detailed functional studies of alternative splicing.
• The findings highlight the critical role of specific RNA isoforms in cellular processes like cell-cycle regulation.