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

Chunlei Jiao, Natalia L. Peeck, Jiaqi Yu, Mohammad Ghaem Maghami, Sarah Kono, Daphne Collias, Sandra L. Martinez Diaz, Rachael Larose and Chase L. Beisel ()
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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)

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

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-50243-x

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