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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number1937
Number of pages12
JournalNature Communications
Volume13
Issue number1
DOIs
Publication statusPublished - 11 Apr 2022

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