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bioRxiv · 10.1101/2021.05.24.445356

Reprogrammed tracrRNAs enable repurposing RNAs as crRNAs and detecting RNAs

Liu, Y.·Pinto, F.·Wan, X.·Peng, S.·Li, M.·Xie, Z.·French, C. E.·Wang, B.
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Abstract

In type II CRISPR systems, the guide RNA (gRNA) consists of a CRISPR RNA (crRNA) and a hybridized trans-acting CRISPR RNA (tracrRNA) which interacts directly with Cas9 and is essential to its guided DNA targeting function. Though tracrRNAs are diverse in sequences and structures across type II CRISPR systems, the programmability of crRNA-tracrRNA hybridization for particular Cas9 has not been studied adequately. Here, we revealed the high programmability of crRNA-tracrRNA hybridization for Streptococcus pyogenes Cas9. By reprogramming the crRNA-tracrRNA hybridized sequence, reprogrammed tracrRNAs can repurpose various RNAs as crRNAs to trigger CRISPR function. We showed that the engineered crRNA-tracrRNA pairs enable design of orthogonal cellular computing devices and hijacking of endogenous RNAs as crRNAs. We next designed novel RNA sensors that can monitor the transcriptional activity of specific genes on the host genome and detect SARS-CoV-2 RNA in vitro. The engineering potential of crRNA-tracrRNA interaction has therefore redefined the capabilities of CRISPR/Cas9 system.

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Liu, Y., Pinto, F., Wan, X., Peng, S., Li, M., Xie, Z., French, C. E., Wang, B.. 2021-05-24. Reprogrammed tracrRNAs enable repurposing RNAs as crRNAs and detecting RNAs. https://doi.org/10.1101/2021.05.24.445356

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