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Programmable RNA detection with CRISPR-Cas12a

Author

Listed:
  • Santosh R. Rananaware

    (University of Florida)

  • Emma K. Vesco

    (University of Florida
    Baylor College of Medicine)

  • Grace M. Shoemaker

    (University of Florida)

  • Swapnil S. Anekar

    (University of Florida)

  • Luke Samuel W. Sandoval

    (University of Florida)

  • Katelyn S. Meister

    (University of Florida)

  • Nicolas C. Macaluso

    (University of Florida)

  • Long T. Nguyen

    (University of Florida)

  • Piyush K. Jain

    (University of Florida
    University of Florida
    University of Florida)

Abstract

Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.

Suggested Citation

  • Santosh R. Rananaware & Emma K. Vesco & Grace M. Shoemaker & Swapnil S. Anekar & Luke Samuel W. Sandoval & Katelyn S. Meister & Nicolas C. Macaluso & Long T. Nguyen & Piyush K. Jain, 2023. "Programmable RNA detection with CRISPR-Cas12a," Nature Communications, Nature, vol. 14(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-41006-1
    DOI: 10.1038/s41467-023-41006-1
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    References listed on IDEAS

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    1. Ines Fonfara & Hagen Richter & Majda Bratovič & Anaïs Le Rhun & Emmanuelle Charpentier, 2016. "The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA," Nature, Nature, vol. 532(7600), pages 517-521, April.
    2. Kean Hean Ooi & Mengying Mandy Liu & Jie Wen Douglas Tay & Seok Yee Teo & Pornchai Kaewsapsak & Shengyang Jin & Chun Kiat Lee & Jingwen Hou & Sebastian Maurer-Stroh & Weisi Lin & Benedict Yan & Gabrie, 2021. "An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing," Nature Communications, Nature, vol. 12(1), pages 1-23, December.
    3. Yongmoon Jeon & You Hee Choi & Yunsu Jang & Jihyeon Yu & Jiyoung Goo & Gyejun Lee & You Kyeong Jeong & Seung Hwan Lee & In-San Kim & Jin-Soo Kim & Cherlhyun Jeong & Sanghwa Lee & Sangsu Bae, 2018. "Direct observation of DNA target searching and cleavage by CRISPR-Cas12a," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
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    Cited by:

    1. Yichuan Chen & Xinping Wang & Junqi Zhang & Qingyuan Jiang & Bin Qiao & Baoxia He & Wenhao Yin & Jie Qiao & Yi Liu, 2024. "Split crRNA with CRISPR-Cas12a enabling highly sensitive and multiplexed detection of RNA and DNA," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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