Polyvalent guide RNAs for CRISPR antivirals.

Area of Science:

• Molecular Biology
• Biotechnology
• Antiviral Research

Background:

• CRISPR effector Cas13 targets and degrades RNA complementary to its guide RNA (gRNA).
• Cas13 shows promise for antiviral biotechnology by degrading viral mRNA and RNA genomes.
• Multiplexed targeting is crucial for effective viral suppression.

Purpose of the Study:

• To develop a strategy for designing gRNAs that maximize activity against multiple viral targets simultaneously.
• To exploit the molecular biophysics of Cas13's promiscuous target recognition for enhanced antiviral efficacy.

Main Methods:

• Designed 'polyvalent' guide RNA sequences (pgRNAs) leveraging Cas13's target recognition properties.
• Tested pgRNA efficacy for multiplexed targeting of viral RNA in *Nicotiana benthamiana*.
• Compared antiviral activity of pgRNAs against conventionally-designed gRNAs.

Main Results:

• pgRNAs achieved superior antiviral elimination at tissue/organ scales in *N. benthamiana* compared to conventional gRNAs.
• pgRNAs reduced detectable viral RNA by >30-fold, even without perfect complementarity to targets.
• Multiplexed pgRNAs reduced viral RNA by >99.5%.

Conclusions:

• Polyvalent gRNAs offer a powerful strategy for enhanced, multiplexed CRISPR-based antiviral targeting.
• This approach significantly improves viral RNA degradation, even with imperfect target complementarity.
• The abundance of pgRNA-targetable sequences in viral genomes underscores the need for specialized CRISPR strategies in eukaryotes.