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Peer-reviewedCC BY-NC-NDNature Biotechnology

DNA-guided CRISPR–Cas12 for cellular RNA targeting

  • Carlos Orosco1
  • Boyu Huang1
  • Santosh R. Rananaware2,1
  • August P. Bodin1
  • Ivy Browning2
  • Anne Fang3
  • Michael P. Baugh1
  • Ian H. Lange1
  • Yasmin B. Elhabashy1
  • Mahika Balaraju4
  • Jordan G. Lewis1
  • Nayan H. Shah5
5 more authors
  • Michael P. Hanna1
  • Sarah J. Flannery1
  • Katelyn S. Meister6
  • Vedant N. Karalkar7
  • Piyush Jain8,9,10
10 affiliations
  1. 1Department of Chemical Engineering, University of Florida, Gainesville, FL, USA
  2. 2CasNx, LLC, Alachua, FL, USA
  3. 3Department of Chemical Biology, University of Florida, Gainesville, FL, USA
  4. 4Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA
  5. 5Department of Microbiology and Cell Sciences, University of Florida, Gainesville, FL, USA
  6. 6Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
  7. 7Department of Computer Science, University of Florida, Gainesville, FL, USA
  8. 8Department of Chemical Engineering, University of Florida, Gainesville, FL, USA. [email protected]
  9. 9Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA. [email protected]
  10. 10UF Health Cancer Center, University of Florida, Gainesville, FL, USA. [email protected]
Open at sourceDOICite

Abstract

Here, we present ΨDNA, a DNA-based guide that enables RNA targeting by Cas12 nucleases, overcoming the traditional reliance on RNA-guided systems. We engineer ΨDNA to mimic a CRISPR RNA (crRNA) scaffold in reverse orientation, allowing AsCas12a and Cas12i1 to recognize RNA and trigger strong single-stranded DNA trans-cleavage for sensitive detection of diverse RNA species, including 100% accurate hepatitis C virus RNA detection in clinical samples. ΨDNA also achieves 70-95% multiplex knockdown of endogenous intracellular RNA transcripts through ribosome stalling across multiple human cell lines. Mechanistic studies reveal that activity depends on a stem loop that stabilizes a catalytically competent Cas12-ΨDNA-RNA complex. Lastly, codelivery of crRNA and ΨDNA enables simultaneous DNA editing and RNA knockdown with a single effector and modular fusions of different enzymes to AsCas12a extend ΨDNA to RNase H-mediated RNA degradation and METTL3-based epitranscriptomic editing. Together, ΨDNA guides constitute an adaptable toolkit that extends Cas12 systems beyond genome editing and diagnostics to enable precise, programmable control of cellular transcriptomes and their epitranscriptomic marks.

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Cite

BibTeXRIS
BibTeX
@article{orosco2026dnaguided,
  title = {{DNA-guided CRISPR–Cas12 for cellular RNA targeting}},
  author = {Carlos Orosco and Boyu Huang and Santosh R. Rananaware and August P. Bodin and Ivy Browning and Anne Fang and Michael P. Baugh and Ian H. Lange and Yasmin B. Elhabashy and Mahika Balaraju and Jordan G. Lewis and Nayan H. Shah and Michael P. Hanna and Sarah J. Flannery and Katelyn S. Meister and Vedant N. Karalkar and Piyush Jain},
  journal = {Nature Biotechnology},
  year = {2026},
  month = may,
  doi = {10.1038/s41587-026-03129-w},
  url = {https://doi.org/10.1038/s41587-026-03129-w},
  note = {CC BY-NC-ND},
}
RIS
TY  - JOUR
TI  - DNA-guided CRISPR–Cas12 for cellular RNA targeting
AU  - Carlos Orosco
AU  - Boyu Huang
AU  - Santosh R. Rananaware
AU  - August P. Bodin
AU  - Ivy Browning
AU  - Anne Fang
AU  - Michael P. Baugh
AU  - Ian H. Lange
AU  - Yasmin B. Elhabashy
AU  - Mahika Balaraju
AU  - Jordan G. Lewis
AU  - Nayan H. Shah
AU  - Michael P. Hanna
AU  - Sarah J. Flannery
AU  - Katelyn S. Meister
AU  - Vedant N. Karalkar
AU  - Piyush Jain
PY  - 2026
DA  - 2026/05/15
JO  - Nature Biotechnology
DO  - 10.1038/s41587-026-03129-w
UR  - https://doi.org/10.1038/s41587-026-03129-w
AB  - Here, we present ΨDNA, a DNA-based guide that enables RNA targeting by Cas12 nucleases, overcoming the traditional reliance on RNA-guided systems. We engineer ΨDNA to mimic a CRISPR RNA (crRNA) scaffold in reverse orientation, allowing AsCas12a and Cas12i1 to recognize RNA and trigger strong single-stranded DNA trans-cleavage for sensitive detection of diverse RNA species, including 100% accurate hepatitis C virus RNA detection in clinical samples. ΨDNA also achieves 70-95% multiplex knockdown of endogenous intracellular RNA transcripts through ribosome stalling across multiple human cell lines. Mechanistic studies reveal that activity depends on a stem loop that stabilizes a catalytically competent Cas12-ΨDNA-RNA complex. Lastly, codelivery of crRNA and ΨDNA enables simultaneous DNA editing and RNA knockdown with a single effector and modular fusions of different enzymes to AsCas12a extend ΨDNA to RNase H-mediated RNA degradation and METTL3-based epitranscriptomic editing. Together, ΨDNA guides constitute an adaptable toolkit that extends Cas12 systems beyond genome editing and diagnostics to enable precise, programmable control of cellular transcriptomes and their epitranscriptomic marks.
SN  - 0733-222X
SN  - 1087-0156
SN  - 1546-1696
SN  - 2331-3684
ER  -