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

Yang Liu; Filipe Pinto; Xinyi Wan; Zhugen Yang; Shuguang Peng; Mengxi Li; Jonathan M Cooper; Zhen Xie; Christopher E French; Baojun Wang

doi:10.1038/s41467-022-29604-x

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

Nat Commun. 2022 Apr 11;13(1):1937. doi: 10.1038/s41467-022-29604-x.

Authors

Yang Liu^{1

2}, Filipe Pinto^#², Xinyi Wan^#², Zhugen Yang^{3

4}, Shuguang Peng⁵, Mengxi Li², Jonathan M Cooper⁶, Zhen Xie⁵, Christopher E French^{2

7}, Baojun Wang^{8

9

10

11}

Affiliations

¹ College of Chemical and Biological Engineering & Hangzhou Innovation Center, Zhejiang University, Hangzhou, 311200, China.
² Centre for Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3FF, UK.
³ Research Centre for Biological Computation, Zhejiang Laboratory, Hangzhou, 311100, China.
⁴ Cranfield Water Science Institute, School of Water, Environment and Energy, Cranfield University, Cranfield, MK43 0AL, UK.
⁵ Center for Synthetic and System Biology, Department of Automation, Beijing National Research Centre for Information Science and Technology, Tsinghua University, Beijing, 100084, China.
⁶ Division of Biomedical Engineering, James Watt School of Engineering, University of Glasgow, Glasgow, G12 8QQ, UK.
⁷ Zhejiang University-University of Edinburgh Joint Research Centre for Engineering Biology, Zhejiang University International Campus, Haining, 314400, China.
⁸ College of Chemical and Biological Engineering & Hangzhou Innovation Center, Zhejiang University, Hangzhou, 311200, China. baojun.wang@zju.edu.cn.
⁹ Centre for Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, EH9 3FF, UK. baojun.wang@zju.edu.cn.
¹⁰ Research Centre for Biological Computation, Zhejiang Laboratory, Hangzhou, 311100, China. baojun.wang@zju.edu.cn.
¹¹ Zhejiang University-University of Edinburgh Joint Research Centre for Engineering Biology, Zhejiang University International Campus, Haining, 314400, China. baojun.wang@zju.edu.cn.

^# Contributed equally.

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.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

Biosensing Techniques*
COVID-19*
CRISPR-Cas Systems / genetics
Humans
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism
RNA, Viral / genetics
SARS-CoV-2 / genetics

Substances

RNA, Guide, CRISPR-Cas Systems
RNA, Viral

Abstract

Publication types

MeSH terms

Substances

Grants and funding