Noncanonical crRNAs derived from host transcripts enable multiplexable RNA detection by Cas9

CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of noncanonical crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp(614)-> Gly) variant with single-base resolution in patient samples.

Automated summary

The study found that tracrRNA hybridizing with cellular RNAs can lead to the formation of new crRNAs capable of guiding DNA targeting by Cas9. Reprogrammed tracrRNAs linking the presence of RNA of interest to DNA targeting were used in the LEOPARD diagnostic platform, allowing simultaneous detection of RNAs from different viruses in one test.