RNA-coupled CRISPR screens reveal ZNF207 as a regulator of LMNA aberrant splicing in progeria

Abstract

Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-seq (CRISPR-based identification of regulators of alternative splicing with phenotypic sequencing), a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitatively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-seq identified both known and untested regulators, enriched for proteins involved in RNA splicing and metabolism. As a proof-of-concept, CRASP-seq analysis of the LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin protein levels in patient-derived cells. We further show that ZNF207’s zinc-finger domain broadly impacts alternative splicing through direct interactions with U1 small nuclear ribonucleoprotein (snRNP) components. 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. • 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 • Depletion of ZNF207 corrects the aberrant LMNA splicing underlying progeria This study introduces CRASP-seq, an RNA-coupled CRISPR screening platform designed to systematically identify regulatory factors and domains of alternative pre-mRNA splicing. Using this technology, ZNF207 is identified as a bona fide splicing regulator through its interaction with U1 snRNP components. ZNF207 modulates aberrant LMNA splicing, a critical event linked to progeria.