Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific SARS-CoV-2 Diagnostic

Santiago-Frangos, Andrew; Hall, Laina N.; Nemudraia, Anna; Nemudryi, Artem; Krishna, Pushya; Wiegand, Tanner; Wilkinson, Royce A.; Snyder, Deann T.; Hedges, Jodi F.; Cicha, Calvin; Lee, Helen H.; Graham, Ava; Jutila, Mark A.; Taylor, Matthew P.; Wiedenheft, Blake

There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here we repurposed the type III CRISPR-Cas system for sensitive and sequence specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design 10 guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/μL for a single guide RNA, to 106 copies/μL for 10 guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μl and is completed using patient samples in less than 30 minutes.