Next Up Pissants - CRISPR crRNA Therapeutic "Vaccinations"

nature |  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.

The remarkable capability of RNA viruses to adapt to selective host and environmental pressure is highly dependent on their ability to generate genomic diversity through the occurrence of de novo mutations⁴⁶. Mutation-driven viral evolution can generate drug resistance, immune escape, and increased efficiency of transmission and pathogenicity, all of which are detrimental to the host. Although our understanding of SARS-CoV-2 mutation-driven escape mechanisms remains limited, the emergence of new variants, which possess increased infective potential⁸ or are resistant to recombinant monoclonal antibodies and antibodies in the sera of convalescent patients and vaccinated individuals^7,8,17,18,36 are of major global concern. In this study, we leveraged an innovative CRISPR-pspCas13b technology and employed two key strategies to silence SARS-CoV-2 RNA and counteract its intrinsic ability to escape standard therapies through the generation of de novo mutations.