Engineered CRISPR/Cas12 Systems Enable Point-of-Care Detection of Sars-Cov-2 RNA

There is an urgent need for a reliable point-of-care (POC) test that is rapid, sensitive, specific, inexpensive, easy-to-use, and accessible for detecting SARS-CoV-2 RNA. While isothermal amplification-based RT-LAMP tests have been authorized by the FDA under the EUA for POC testing, these tests have poor specificity and cannot be easily developed to discriminate between different variants of concern (VOC) of SARS-CoV-2. Most Class 2 CRISPR/Cas systems, including CRISPR/Cas12 and CRISPR/Cas 13 mediate a nonspecific collateral trans-cleavage of random DNA and RNA after binding or cis-cleavage of their target DNA or RNA. This trans-cleavage activity has been widely exploited for nucleic acid detection, including SARS-CoV-2 RNA. Both Cas13a-based and Cas12a-based assays have been assigned EUA by the FDA for testing SARS-CoV-2 RNA in a high-complexity lab environment, however, FDA has not yet authorized a CRISPR-based POC test for SARS-CoV-2.

We discovered that crRNAs of Cas12a can tolerate various length extensions with DNA or RNA on both 3’- and 5’-ends (Nat. Comms., 2020). In particular, a crRNA with 7-nt DNA extensions on its 3’-end, we named CRISPR-ENHANCE, augmented the rate of Cas12a-mediated trans-cleavage activity by 3.2-fold. By combining with a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) step, a single copy of SARS-CoV-2 RNA could be detected using a lateral flow assay within 50 minutes (Methods, 2021). Compared to the wild-type crRNAs, our CRISPR-ENHANCE system demonstrated up to 5-fold higher sensitivity in detecting SARS-CoV-2 RNA. Our CRISPR-ENHANCE-based COVID-19 tests demonstrated high inclusivity and exclusivity and achieved 96.7% sensitivity and 96.7% specificity in clinical samples and a lyophilized version was stable for at least 30 days at room temperature (medRxiv, 2021; Comms. Med., in rev.). We also purified 22 orthologs of Cas12a from different species, and for the first time, combinatorially tested them with 9 crRNA orthologs over 198 combinations and identified several new functional combinations of crRNA/Cas12a (cCRISPR) for SARS-CoV-2 alpha (B.1.1.7) VOC (medRxiv, 2021; Sci. Adv., in rev.). Most recently, we combined an ultra-thermo-stable CRISPR/Cas12b with RT-LAMP to create a single-pot reaction that can monitor the RT-LAMP-based amplification and CRISPR-cleavage using a dual-color fluorescence test detecting SARS-CoV-2 VOC within 10-20 minutes in saliva.