Polyvalent guide RNAs for CRISPR antivirals

CRISPR effector Cas13 recognizes and degrades RNA molecules that are complementary to its guide RNA (gRNA) and possesses potential as an antiviral biotechnology because it can degrade viralmRNAandRNAgenomes. Because multiplexed targeting is a critical strategy to improve viral suppression, we developed a strategy to design of gRNAs where individual gRNAs have maximized activity at multiple viral targets, simultaneously, by exploiting the molecular biophysics of promiscuous target recognition by Cas13. These ``polyvalent'' gRNA sequences (``pgRNAs'') provide superior antiviral elimination across tissue/organ scales in a higher organism (Nicotiana benthamiana) compared to conventionally-designed gRNAs-reducing detectable viral RNA by >30-fold, despite lacking perfect complementarity with either of their targets and, when multiplexed, reducing viral RNA by >99.5%. Pairs of pgRNA-targetable sequences are abundant in the genomes of RNA viruses, and this work highlights the need for specific approaches to the challenges of targeting viruses in eukaryotes using CRISPR.

Automated summary

This study developed a strategy to design polyvalent gRNAs that can simultaneously target multiple viral sites with high activity, leading to superior antiviral elimination in higher organisms.