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Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors

Author

Listed:
  • Yang Liu

    (Zhejiang University
    University of Edinburgh)

  • Filipe Pinto

    (University of Edinburgh)

  • Xinyi Wan

    (University of Edinburgh)

  • Zhugen Yang

    (Research Centre for Biological Computation, Zhejiang Laboratory
    Cranfield University)

  • Shuguang Peng

    (Tsinghua University)

  • Mengxi Li

    (University of Edinburgh)

  • Jonathan M. Cooper

    (University of Glasgow)

  • Zhen Xie

    (Tsinghua University)

  • Christopher E. French

    (University of Edinburgh
    Zhejiang University International Campus)

  • Baojun Wang

    (Zhejiang University
    University of Edinburgh
    Research Centre for Biological Computation, Zhejiang Laboratory
    Zhejiang University International Campus)

Abstract

In type II CRISPR systems, the guide RNA (gRNA) comprises a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA), both being essential in guided DNA targeting functions. Although tracrRNAs are diverse in sequence and structure across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for Cas9 is not fully understood. Here, we reveal the programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9, and in doing so, redefine the capabilities of Cas9 proteins and the sources of crRNAs, providing new biosensing applications for type II CRISPR systems. By reprogramming the crRNA-tracrRNA hybridized sequence, we show that engineered crRNA-tracrRNA interactions can not only enable the design of orthogonal cellular computing devices but also facilitate the hijacking of endogenous small RNAs/mRNAs as crRNAs. We subsequently describe how these re-engineered gRNA pairings can be implemented as RNA sensors, capable of monitoring the transcriptional activity of various environment-responsive genomic genes, or detecting SARS-CoV-2 RNA in vitro, as an Atypical gRNA-activated Transcription Halting Alarm (AGATHA) biosensor.

Suggested Citation

  • Yang Liu & Filipe Pinto & Xinyi Wan & Zhugen Yang & Shuguang Peng & Mengxi Li & Jonathan M. Cooper & Zhen Xie & Christopher E. French & Baojun Wang, 2022. "Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-29604-x
    DOI: 10.1038/s41467-022-29604-x
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    References listed on IDEAS

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