tetano
Editor, Senior Moderator
Nat Commun
. 2021 Jul 13;12(1):4270.
doi: 10.1038/s41467-021-24577-9.
Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance
Mohamed Fareh[SUP] 1 2 [/SUP], Wei Zhao[SUP] 3 [/SUP], Wenxin Hu[SUP] 4 5 [/SUP], Joshua M L Casan[SUP] 4 5 [/SUP], Amit Kumar[SUP] 4 5 [/SUP], Jori Symons[SUP] 3 [/SUP], Jennifer M Zerbato[SUP] 3 [/SUP], Danielle Fong[SUP] 3 [/SUP], Ilia Voskoboinik[SUP] 4 5 [/SUP], Paul G Ekert[SUP] 4 5 6 7 [/SUP], Rajeev Rudraraju[SUP] 3 8 9 [/SUP], Damian F J Purcell[SUP] 9 [/SUP], Sharon R Lewin[SUP] #[/SUP][SUP] 10 11 12 [/SUP], Joseph A Trapani[SUP] #[/SUP][SUP] 4 5 [/SUP]
Affiliations
Abstract
The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide 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 Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.
. 2021 Jul 13;12(1):4270.
doi: 10.1038/s41467-021-24577-9.
Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance
Mohamed Fareh[SUP] 1 2 [/SUP], Wei Zhao[SUP] 3 [/SUP], Wenxin Hu[SUP] 4 5 [/SUP], Joshua M L Casan[SUP] 4 5 [/SUP], Amit Kumar[SUP] 4 5 [/SUP], Jori Symons[SUP] 3 [/SUP], Jennifer M Zerbato[SUP] 3 [/SUP], Danielle Fong[SUP] 3 [/SUP], Ilia Voskoboinik[SUP] 4 5 [/SUP], Paul G Ekert[SUP] 4 5 6 7 [/SUP], Rajeev Rudraraju[SUP] 3 8 9 [/SUP], Damian F J Purcell[SUP] 9 [/SUP], Sharon R Lewin[SUP] #[/SUP][SUP] 10 11 12 [/SUP], Joseph A Trapani[SUP] #[/SUP][SUP] 4 5 [/SUP]
Affiliations
- PMID: 34257311
- DOI: 10.1038/s41467-021-24577-9
Abstract
The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide 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 Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.