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bioRxiv · 10.1101/2020.11.18.389312

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

Fareh, M.·Zhao, W.·Hu, W.·Casan, J. M.·Kumar, A.·Symons, J.·Voskoboinik, I.·Ekert, P. G.·Rudraraju, R.·Lewin, S. R.·Trapani, J. A.
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Abstract

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.

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Fareh, M., Zhao, W., Hu, W., Casan, J. M., Kumar, A., Symons, J., Voskoboinik, I., Ekert, P. G., Rudraraju, R., Lewin, S. R., Trapani, J. A.. 2020-11-19. Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance. https://doi.org/10.1101/2020.11.18.389312

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