Summary CRISPR /Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR /Cas effector Cas13a is an RNA ‐targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR /Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y ( PVY ). Transgenic potato lines expressing Cas13a/sg RNA (small guide RNA ) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sg RNA construct in the plants. Our data further demonstrate that appropriately designed sg RNA s can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S . Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.

CRISPR-Cas systems endow the bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However, whether gRNA targeting sites have effects on the efficiency of RNA viruses inhibition is unknown. Here we repurpose of CRISPR/Cas13a coupled with an endogenous tRNA-processing system (polycistronic tRNA-gRNA, PTG) to target four genes of potato virus Y (PVY). Expression of Cas13a and four different gRNAs were evidenced in transgenic potato lines expressing Cas13a/PTG construct. We show that the multiple gRNAs transgenic plants could suppress PVY accumulation and disease symptoms at a similar level to transgenic plants expressing single gRNA. Collectively, our study suggests that the Cas13-based multiplex RNA targeting system can be used to engineer resistances to RNA viruses in plants, and the number of gRNAs target sites have no effect on CRISPR/Cas13a-mediated viral interference in plants.