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TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases

Author

Listed:
  • Chunlei Jiao

    (Helmholtz-Centre for Infection Research (HZI))

  • Natalia L. Peeck

    (Helmholtz-Centre for Infection Research (HZI))

  • Jiaqi Yu

    (Helmholtz-Centre for Infection Research (HZI))

  • Mohammad Ghaem Maghami

    (Helmholtz-Centre for Infection Research (HZI))

  • Sarah Kono

    (Helmholtz-Centre for Infection Research (HZI))

  • Daphne Collias

    (Helmholtz-Centre for Infection Research (HZI))

  • Sandra L. Martinez Diaz

    (Helmholtz-Centre for Infection Research (HZI))

  • Rachael Larose

    (Helmholtz-Centre for Infection Research (HZI))

  • Chase L. Beisel

    (Helmholtz-Centre for Infection Research (HZI)
    University of Würzburg)

Abstract

Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA’s presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage—all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.

Suggested Citation

  • Chunlei Jiao & Natalia L. Peeck & Jiaqi Yu & Mohammad Ghaem Maghami & Sarah Kono & Daphne Collias & Sandra L. Martinez Diaz & Rachael Larose & Chase L. Beisel, 2024. "TracrRNA reprogramming enables direct PAM-independent detection of RNA with diverse DNA-targeting Cas12 nucleases," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-50243-x
    DOI: 10.1038/s41467-024-50243-x
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