Deep screening of proximal and distal splicing-regulatory elements in a native sequence context

Abstract

Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is impeded by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically dead CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied toSMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identified not only known SREs, but also a novel distal intronic splicing enhancer, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.

Engineered RNA‐Binding Proteins: Studying and Controlling RNA Regulation

“…Recinos et al validated this strategy on the well-studied SMN2 transcript, whose alternative splicing is a promising therapeutic target for spinal muscular atrophy. [90] Their pooled screen not only identified well-known SREs controlling SMN2 splicing, but also a novel distal intronic element far outside previous screens, that impacted exon 7 inclusion. These results were exciting because intronic distal elements have been reported before but are difficult to identify de novo due to their undefined reliance on surrounding sequence context and relative distance from the affected exon.…”