Citation
- Authors: Yu D . et al.
- Year: 2023
- Journal: Mol Ther S1525-0016 00139-9
- Applications: in vitro / crRNA, dsRNA, mRNA / jetMESSENGER
- Cell types:
- Name: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T - Name: VERO E6
- Name: HEK-293T
Abstract
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.