The COVID-19 pandemic has emerged as a catastrophic global emergency. 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. In this study, we employed genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs (Fareh et al, Nature Comms, 2021). We show that Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13 CRISPR RNAs (crRNAs) markedly suppressed viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do 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.
Mohamed Fareh is a senior research fellow with a multidisciplinary background in RNA biology, CRISPR, cancer, and advanced single-molecule biophysics. He obtained his Ph.D. in 2012 (France), where he uncovered a subset of non-coding RNAs that regulate cell plasticity in high-grade brain tumours. He did his postdoctoral training in the single-molecule Biophysics lab (Joo lab, TU Delft, The Netherlands), where he developed new single-molecule approaches to investigate the biology of non-coding RNAs and CRISPR effectors with high spatial and temporal resolution. In August 2018, Mohamed relocated to Australia to join the Trapani lab at the Peter MacCallum Cancer Centre (Melbourne). He leads projects focusing on reprogramming RNA-targeting CRISPR tools called CRISPR-Cas13 for precise transcriptome remodelling. His team is interested in (i) understanding the molecular basis of RNA recognition by CRISPR-Cas13, and (ii) reprogramming CRISPR-Cas13 to silence undruggable tumour drivers and viral transcripts.