Department of Nanoscience, The University of North Carolina at Greensboro, Greensboro, NC 27401, USA
Rachel Tinker-Kulberg
Department of Nanoscience, The University of North Carolina at Greensboro, Greensboro, NC 27401, USA
Mohammad Salehin
Department of Nanoscience, The University of North Carolina at Greensboro, Greensboro, NC 27401, USA
Tinku Supakar
Department of Nanoscience, The University of North Carolina at Greensboro, Greensboro, NC 27401, USA
Sydney Chamberlain
Department of Biology, The University of North Carolina at Greensboro, Greensboro, NC 27401, USA
Ayalew Ligaba-Osena
Department of Biology, The University of North Carolina at Greensboro, Greensboro, NC 27401, USA
Eric A. Josephs
Department of Nanoscience, The University of North Carolina at Greensboro, Greensboro, NC 27401, USA; Department of Biology, The University of North Carolina at Greensboro, Greensboro, NC 27401, USA; Corresponding author
Summary: 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 viral mRNA and RNA genomes. 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.
