CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements

Yocelyn Recinos, Dmytro Ustianenko, Yow-Tyng Yeh, Xiaojian Wang, Martin Jacko, Lekha V. Yesantharao, Qiyang Wu and Chaolin Zhang ()
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Yocelyn Recinos: Columbia University
Dmytro Ustianenko: Columbia University
Yow-Tyng Yeh: Columbia University
Xiaojian Wang: Columbia University
Martin Jacko: Columbia University
Lekha V. Yesantharao: Columbia University
Qiyang Wu: Columbia University
Chaolin Zhang: Columbia University

Nature Communications, 2024, vol. 15, issue 1, 1-15

Abstract: 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 hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically 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 identifies not only known SREs but also a previously unknown distal intronic SRE, 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.

Date: 2024
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DOI: 10.1038/s41467-024-47140-8

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