Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance

• Published in: Nature communications Vol. 12; no. 1; pp. 1 - 16
• Main Authors: Fareh, Mohamed, Zhao, Wei, Hu, Wenxin, Casan, Joshua M. L., Kumar, Amit, Symons, Jori, Zerbato, Jennifer M., Fong, Danielle, Voskoboinik, Ilia, Ekert, Paul G., Rudraraju, Rajeev, Purcell, Damian F. J., Lewin, Sharon R., Trapani, Joseph A.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 13-07-2021; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/1; 13/106; 13/109; 13/31; 13/44; 13/89; 14; 14/35; 14/63; 38; 38/22; 38/44; 38/70; 42; 45; 49; 631/326/4041; 631/337/505; 631/61/338/552; 631/61/391/3932; 82/29; Computer applications; Coronaviruses; CRISPR; Drug development; Humanities and Social Sciences; Mammalian cells; Mammals; multidisciplinary; Mutagenesis; Mutants; Nucleocapsids; Nucleotides; Replication; Science; Science (multidisciplinary); Severe acute respiratory syndrome; Severe acute respiratory syndrome coronavirus 2; Spikes; Viral diseases; Viruses
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-24577-9

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.
Cas13b can be harnessed to target and degrade RNA transcripts inside a cellular environment. Here the authors reprogram Cas13b to target SARSCoV-2 transcripts in infected mammalian cells and reveal its resilience to variants thanks to single mismatch tolerance.