Pseudoknot-targeting Cas13b combats SARS-CoV-2 infection by suppressing viral replication

Area of Science:

• Molecular Biology
• Virology
• Gene Editing

Background:

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a significant global health threat.
• CRISPR-Cas13 systems offer a novel RNA-targeting strategy for antiviral defense.
• Targeting conserved viral regions is crucial for broad-spectrum efficacy.

Purpose of the Study:

• To develop and evaluate a CRISPR-Cas13b system for targeting and degrading SARS-CoV-2 RNA.
• To identify optimal CRISPR RNA (crRNA) targets within the SARS-CoV-2 genome for maximal antiviral effect.
• To assess the therapeutic efficacy of the developed system in a preclinical mouse model.

Main Methods:

• Designed mRNA-encoded Cas13b and 12 distinct CRISPR RNAs (crRNAs) targeting the SARS-CoV-2 open reading frame 1b (ORF1b) region.
• Evaluated crRNA efficacy in suppressing viral propagation and gene expression.
• Quantified viral load reduction and spike protein expression.
• Validated therapeutic potential in hACE2 transgenic mice infected with SARS-CoV-2.

Main Results:

• crRNAs targeting the pseudoknot site upstream of ORF1b demonstrated the highest efficacy in inhibiting viral replication.
• Pseudoknot-targeting Cas13b reduced SARS-CoV-2 spike protein expression and viral replication by 99%.
• The system exhibited broad potency, inhibiting replication of multiple SARS-CoV-2 variants.
• In vivo studies in mice showed significant reduction in viral titers following crRNA treatment.

Conclusions:

• The pseudoknot region represents a strategic target for effective genomic degradation of SARS-CoV-2.
• Pseudoknot-targeting Cas13b demonstrates significant potential as a breakthrough therapeutic strategy against SARS-CoV-2.
• This approach may be applicable for treating infections caused by other RNA viruses.