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

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 hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically dead inactive 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 to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identified not only known SREs, but also a novel distal intronic splicing enhancerelement, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs.