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.
Highlights
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
The comprehensive mutagenesis analysis in this study further demonstrate that a single Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNAs (crRNAs) is resilient to single-nucleotide mismatch and remain catalytically active against mutated SARS-CoV-2 RNAs that arise in new variants
While NT crRNA exhibited no effect on enhanced green fluorescent protein (eGFP) expression, the Spike-targeting crRNA2 showed dosedependent silencing of the Spike transcript with 50% silencing efficiency (IC50) achieved with 5.16 pM of plasmid crRNA, demonstrating that crRNA availability in the cell is key for efficient degradation of viral RNA (Fig. 1d, e)
Summary
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. The single nucleotide substitution (A to G) in the receptor binding domain of the Spike glycoprotein, demonstrated increased affinity for the ACE2 receptor and enhanced replication capacity in vitro, potentially contributing to the global spread of this variant[8,9,10,11] This was followed by the B.1.1.7 strain, which is the global dominant variant of concern[12]. The comprehensive mutagenesis analysis in this study further demonstrate that a single crRNA is resilient to single-nucleotide mismatch and remain catalytically active against mutated SARS-CoV-2 RNAs that arise in new variants These results provide a proof-of-concept for the development of pspCas13b viral suppressors that can circumvent the evolution of SARS-CoV-2 and other pathogenic viruses
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