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

Author

Listed:
  • Yocelyn Recinos

    (Columbia University
    Columbia University)

  • Dmytro Ustianenko

    (Columbia University
    Columbia University
    Flagship Pioneering)

  • Yow-Tyng Yeh

    (Columbia University
    Columbia University)

  • Xiaojian Wang

    (Columbia University
    Columbia University)

  • Martin Jacko

    (Columbia University
    Columbia University
    Inc.)

  • Lekha V. Yesantharao

    (Columbia University
    Columbia University
    Johns Hopkins University School of Medicine)

  • Qiyang Wu

    (Columbia University
    Columbia University)

  • Chaolin Zhang

    (Columbia University
    Columbia University)

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.

Suggested Citation

  • Yocelyn Recinos & Dmytro Ustianenko & Yow-Tyng Yeh & Xiaojian Wang & Martin Jacko & Lekha V. Yesantharao & Qiyang Wu & Chaolin Zhang, 2024. "CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-47140-8
    DOI: 10.1038/s41467-024-47140-8
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