Polyvalent Guide RNAs for CRISPR Antivirals

Abstract

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 higher plants 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 a “monovalent” gRNA counterpart. These results represent a powerful new approach to gRNA design for antiviral applications that can be readily incorporated into current viral detection and therapeutic strategies, and 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.

CRISPR/Cas‐based tools for the targeted control of plant viruses

“…For viral interference applications, these reports are encouraging for future multiplex strategies that can simultaneously either target multiple species of viruses within the same family to provide broad virus protection or target multiple regions of a single virus genome to evade the possibility of evolutionary resistance to the CRISPR/Cas system from occurring. A recent study showed that single polyvalent gRNAs (pgRNAs), designed for one spacer to be able to target multiple viral target sequences, in complex with the CasRx effector can effectively suppress virus spread and gene expression in planta, better than those with a monovalent gRNA counterpart (Bagchi et al, 2022 ). This observation of enhanced antiviral suppression is related to improvements reported by CRISPR antiviral treatments with multiple gRNAs, and future studies could now also use multiple pgRNAs to further increase the number of target sites.…”

Plant genome engineering from lab to field—a Keystone Symposia report

“…Bagchi showed that while monovalent gRNAs only targeted protospacers that resembled their own sequence, the pgRNA was highly active at both sites. In in vivo experiments, pgRNAs were able to robustly suppress TMV spread in N. benthamiana and were more effective than monovalent guides 106 …”