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

Liu, Y., Pinto, F., Wan, X., Yang, Z., Peng, S., Li, M., Cooper, J. M. , Xie, Z., French, C. E. and Wang, B. (2022) Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors. Nature Communications, 13, 1937. (doi: 10.1038/s41467-022-29604-x) (PMID:35410423) (PMCID:PMC9001733)

Abstract

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.

• Item Type: Articles
• Additional Information: his work was supported by the UK Research and Innovation Future Leaders Fellowship [MR/S018875/1], Leverhulme Trust grant [RPG-2020-241], US Office of Naval Research Global grant [N62909-20-1-2036] and Wellcome Trust Institutional Translational Partnership Award. Z.X. was supported by the Natural Science Foundation of China (31771483, 61721003). J.C. was supported by the UK Global Challenges Research Fund through funding from the Engineering and Physical Sciences Research Council (EP/R01437X/1, co-funded by the UK National Institute for Health Research), as well as the UK Medical Research Council (MR/V035401/1).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Cooper, Professor Jonathan
• Authors: Liu, Y., Pinto, F., Wan, X., Yang, Z., Peng, S., Li, M., Cooper, J. M., Xie, Z., French, C. E., and Wang, B.
• College/School: College of Science and Engineering > School of Engineering > Biomedical Engineering
• Journal Name: Nature Communications
• Publisher: Nature Research
• ISSN: 2041-1723
• ISSN (Online): 2041-1723
• Copyright Holders: Copyright © 2022 The Authors
• First Published: First published in Nature Communications 13(1):1937
• Publisher Policy: Reproduced under a Creative Commons Licence

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
300573		Novel low cost diagnostic tools and their impact in Africa	Jonathan Cooper	Engineering and Physical Sciences Research Council (EPSRC)	EP/R01437X/1	ENG - Biomedical Engineering
311995		COVID	Jonathan Cooper	Medical Research Council (MRC)	MR/V035401/1	ENG - Biomedical Engineering