A Scalable, Easy-to-Deploy, Protocol for Cas13-Based Detection of SARS-CoV-2 Genetic Material

Jennifer N Rauch; Eric Valois; Sabrina C Solley; Friederike Braig; Ryan S Lach; Naomi J Baxter; Kenneth S Kosik; Carolina Arias; Diego Acosta-Alvear; Maxwell Z Wilson

To validate a streamlined workflow with these devices, we measured the presence of the three viral sequences that correspond to the CDC RT-qPCR test 19 . Briefly, we PAGE purified annealed synthetic DNA oligonucleotides flanked by an upstream T7 RNA polymerase promoter that encode sequences corresponding to the N1, N2, and N3 sites in the SARS-CoV-2 nucleocapsid gene (Table S3 ). Next, we transcribed the DNA in vitro to obtain target RNAs. After Cas13 purification and extensive optimization of the reaction conditions (Fig. S1 , S2), we used these targets to determine the detection limit of the CREST protocol and found that we could detect up to 10 copies of a target RNA molecule per microliter (Fig. 2B) . This result shows that CREST is as sensitive as the corresponding RT-qPCR in our hands, demonstrating the power of CREST for pairing a thermal cycling amplification step (PCR) with a linear amplification step (transcription), combined with enzymatic signal amplification through fluorescence detection.