Pseudoknot-targeting Cas13b combats SARS- CoV-2 infection viral

CRISPR-Cas13-mediated viral genome targeting is a novel strategy for defending against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Here, we generated mRNA-encoded Cas13b targeting the open reading frame 1b (ORF1b) region to effectively degrade the RNA-dependent RNA polymerase gene. Of the 12 designed CRISPR RNAs (crRNAs), those targeting the pseudoknot site upstream of ORF1b were found to be the most effective in suppressing SARS-CoV-2 propagation. Pseudoknot-targeting Cas13b reduced expression of the spike protein and attenuated viral replication by 99%. It also inhibited the replication of multiple SARS-CoV-2 variants, exhibiting broad potency. We validated the therapeutic efficacy of this system in SARS-CoV-2-infected hACE2 transgenic mice, demonstrating that crRNA treatment significantly reduced viral titers. Our findings suggest that the pseudoknot region is a strategic site for targeted genomic degradation of SARS-CoV-2. Hence, pseudoknot-targeting Cas13b could be a breakthrough therapy for overcoming infections by SARS-CoV-2 or other RNA viruses.

Automated summary

CRISPR-Cas13-mediated viral genome targeting is an effective strategy for combating SARS-CoV-2 and its variants. By using mRNA-encoded Cas13b, specifically targeting the ORF1b region, the RNA-dependent RNA polymerase gene can be degraded. Among the designed CRISPR RNAs, those targeting the pseudoknot site upstream of ORF1b were found to be most effective in suppressing viral propagation. Pseudoknot-targeting Cas13b not only reduced spike protein expression and viral replication by 99%, but also inhibited the replication of multiple SARS-CoV-2 variants.