RNA-coupled CRISPR Screens Reveal ZNF207 as a Regulator of LMNA Aberrant Splicing in Progeria

Abstract

SUMMARY: Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-Seq, a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-Seq identifies both known and novel regulators, enriched for proteins involved in RNA splicing and metabolism. As proof-of-concept, CRASP-Seq analysis of an LMNA cryptic splicing event linked to progeria uncovered Z NF 207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin levels in patient-derived cells. High-throughput mutagenesis further showed that ZNF207’s zinc finger domain broadly impacts alternative splicing through interactions with U1 snRNP factors. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-Seq to uncover key regulators and domains of alternative splicing. Main Points: CRASP-Seq: RNA-coupled CRISPR screen quantifying gene and domain impact on splicing Profiling of five events identified 370 genes influencing alternative splicing ZNF207 regulates splicing by interacting with U1 snRNP via its zinc-finger domains ZNF207 depletion corrects LMNA aberrant splicing causing progeria