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Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing

Author

Listed:
  • David N. Fiflis

    (Duke University)

  • Nicolas A. Rey

    (Duke University School of Medicine)

  • Harshitha Venugopal-Lavanya

    (Duke University)

  • Beatrice Sewell

    (Duke University School of Medicine)

  • Aaron Mitchell-Dick

    (Duke University School of Medicine)

  • Katie N. Clements

    (Duke University School of Medicine)

  • Sydney Milo

    (Duke University)

  • Abigail R. Benkert

    (Duke University School of Medicine)

  • Alan Rosales

    (Duke University)

  • Sophia Fergione

    (Duke University School of Medicine)

  • Aravind Asokan

    (Duke University
    Duke University School of Medicine
    Duke University School of Medicine)

Abstract

Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5’ and 3’ segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts.

Suggested Citation

  • David N. Fiflis & Nicolas A. Rey & Harshitha Venugopal-Lavanya & Beatrice Sewell & Aaron Mitchell-Dick & Katie N. Clements & Sydney Milo & Abigail R. Benkert & Alan Rosales & Sophia Fergione & Aravind, 2024. "Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-46172-4
    DOI: 10.1038/s41467-024-46172-4
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    References listed on IDEAS

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    2. Luke W. Koblan & Michael R. Erdos & Christopher Wilson & Wayne A. Cabral & Jonathan M. Levy & Zheng-Mei Xiong & Urraca L. Tavarez & Lindsay M. Davison & Yantenew G. Gete & Xiaojing Mao & Gregory A. Ne, 2021. "In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice," Nature, Nature, vol. 589(7843), pages 608-614, January.
    3. Bo Zhang & Yangmiao Ye & Weiwei Ye & Vanja Perčulija & Han Jiang & Yiyang Chen & Yu Li & Jing Chen & Jinying Lin & Siqi Wang & Qi Chen & Yu-San Han & Songying Ouyang, 2019. "Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
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