PT  - JOURNAL ARTICLE
AU  - Mohamed Fareh
AU  - Wei Zhao
AU  - Wenxin Hu
AU  - Joshua ML Casan
AU  - Amit Kumar
AU  - Jori Symons
AU  - Ilia Voskoboinik
AU  - Paul G Ekert
AU  - Rajeev Rudraraju
AU  - Sharon R Lewin
AU  - Joseph A Trapani
TI  - Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance
AID  - 10.1101/2020.11.18.389312
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.11.18.389312
4099  - http://biorxiv.org/content/early/2020/11/19/2020.11.18.389312.short
4100  - http://biorxiv.org/content/early/2020/11/19/2020.11.18.389312.full
AB  - Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.Competing Interest StatementCompeting interests. S.R.L is a member of advisory boards of Merck and Gilead and has received investigator-initiated industry funded grants from Merck, Gilead and Viiv. None of this support is relevant to the work in this manuscript. The other authors declare no conflict of interest.