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Massively parallel profiling of RNA-targeting CRISPR-Cas13d

Author

Listed:
  • Hung-Che Kuo

    (University of Texas at Austin)

  • Joshua Prupes

    (University of Texas at Austin)

  • Chia-Wei Chou

    (University of Texas at Austin)

  • Ilya J. Finkelstein

    (University of Texas at Austin
    University of Texas at Austin)

Abstract

CRISPR-Cas13d cleaves RNA and is used in vivo and for diagnostics. However, a systematic understanding of its RNA binding and cleavage specificity is lacking. Here, we describe an RNA Chip-Hybridized Association-Mapping Platform (RNA-CHAMP) for measuring the binding affinity for > 10,000 RNAs containing structural perturbations and other alterations relative to the CRISPR RNA (crRNA). Deep profiling of Cas13d reveals that it does not require a protospacer flanking sequence but is exquisitely sensitive to secondary structure within the target RNA. Cas13d binding is penalized by mismatches in the distal crRNA-target RNA region, while alterations in the proximal region inhibit nuclease activity. A biophysical model built from these data reveals that target recognition initiates in the distal end of the target RNA. Using this model, we design crRNAs that can differentiate between SARS-CoV-2 variants by modulating nuclease activation. This work describes the key determinants of RNA targeting by a type VI CRISPR enzyme.

Suggested Citation

  • Hung-Che Kuo & Joshua Prupes & Chia-Wei Chou & Ilya J. Finkelstein, 2024. "Massively parallel profiling of RNA-targeting CRISPR-Cas13d," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-44738-w
    DOI: 10.1038/s41467-024-44738-w
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    References listed on IDEAS

    as
    1. Alexandra East-Seletsky & Mitchell R. O’Connell & Spencer C. Knight & David Burstein & Jamie H. D. Cate & Robert Tjian & Jennifer A. Doudna, 2016. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection," Nature, Nature, vol. 538(7624), pages 270-273, October.
    2. 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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