Polyvalent Guide RNAs for CRISPR Antivirals

CRISPR biotechnologies, where CRISPR effectors recognize and degrade specific nucleic acid targets that are complementary to their guide RNA (gRNA) cofactors, have been primarily used as a tool for precision gene editing but possess an emerging potential for novel antiviral diagnostics, prophylactics, and therapeutics. In gene editing applications, significant efforts are made to limit the natural tolerance of CRISPR effectors for nucleic acids with imperfect complementarity to their gRNAs in order to prevent degradation and mutation at unintended or “off-target” sites; here we exploit those tolerances to engineer gRNAs that are optimized to promote activity at multiple viral target sites, simultaneously, given that multiplexed targeting is a critical tactic for improving viral detection sensitivity, expanding recognition of clinical strain variants, and suppressing viral mutagenic escape from CRISPR antivirals.

We demonstrate in vitro and in living plants (Nicotiana benthamiana) that single “polyvalent” gRNAs (pgRNAs) in complex with CRISPR effectors Cas9 or Cas13 can effectively degrade pairs of viral targets with significant sequence divergence (up to 40% nucleotide differences) that are prevalent in viral genomes. We find that CRISPR antivirals using pgRNAs can robustly suppress the propagation of plant RNA viruses, in vivo, better than those with “monovalent” gRNA counterparts. Furthermore, we find that the Cas13’s “collateral activity,” which is used in CRISPR-based viral diagnostics, can be robustly activated when in complex with an engineered pgRNAs by multiple segments of the RNA genome of SARS-CoV-2 for sensitive detection of the virus responsible for COVID-19.

Despite the advantages for antiviral applications demonstrated here, “polyvalent” guides would be algorithmically rejected using current computational gRNA design tools for gene editing that prioritize specificity in CRISPR targeting. Our computational design pipeline and experimental validation for pgRNAs in vitro and in vivo demonstrate a powerful new approach to gRNA design for antiviral applications that can be readily incorporated into current viral detection and therapeutic strategies, and they also highlight the need for specific approaches and tools that can address the differential requirements of precision gene editing vs. CRISPR antiviral applications in order to mature these promising biotechnologies.