CRISPR-based RNA detection tools primarily use the RNA-targeting Cas13 system, which detects one target per test. Beisel and colleagues have now reprogrammed the DNA-targeting Cas9 system into a tool that detects multiple RNAs in a single reaction.
In the type II CRISPR system, a trans-activating (tracrRNA) hybridizes to a CRISPR RNA (crRNA), forming a duplex that directs Cas9 to cleave DNA. The authors found that host transcripts outside of the CIRSPR–Cas locus can act as non-canonical crRNAs and pair with the tracrRNA to guide cleavage. Inspired by this unexpected discovery, they designed a Cas9-based RNA detection platform called LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection), consisting of a tracrRNA reprogrammed to pair with an RNA of interest, the Cas9 protein, and a fluorescent reporter containing the DNA target. The presence of the given RNA in the sample licenses Cas9 activity, yielding measurable signals. The authors then used gel electrophoresis or a bioanalyzer to detect distinct cleavage products, thereby enabling multiplex RNA detection. As a proof of concept, they applied LEOPARD to detect multiple viral RNAs in a single test and to distinguish SARS-CoV-2 variants in patient samples. Overall, LEOPARD expands CRISPR diagnostic tools by allowing for multiplex RNA detection.
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Wang, J. Cas9-based RNA detection. Nat Cell Biol 23, 677 (2021). https://doi.org/10.1038/s41556-021-00718-6
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DOI: https://doi.org/10.1038/s41556-021-00718-6