CRISPR/Cas9-mediated viral interference in plants

TLDR

CRISPR/Cas9 confers molecular immunity in microbes and has been adapted for genome editing across eukaryotes. The study aims to determine whether CRISPR/Cas9 can provide plants with molecular immunity against DNA viruses and enable simultaneous targeting of multiple viruses. sgRNAs targeting TYLCV were delivered into Cas9‑expressing *Nicotiana benthamiana* plants, which were then challenged with the virus. CRISPR/Cas9 targeting TYLCV caused viral DNA degradation, induced mutations, and all sgRNAs—particularly those against the origin stem‑loop—reduced viral accumulation and symptoms, proving the system’s efficacy and its capacity to target multiple DNA viruses.

Abstract

The CRISPR/Cas9 system provides bacteria and archaea with molecular immunity against invading phages and conjugative plasmids. Recently, CRISPR/Cas9 has been used for targeted genome editing in diverse eukaryotic species.In this study, we investigate whether the CRISPR/Cas9 system could be used in plants to confer molecular immunity against DNA viruses. We deliver sgRNAs specific for coding and non-coding sequences of tomato yellow leaf curl virus (TYLCV) into Nicotiana benthamiana plants stably overexpressing the Cas9 endonuclease, and subsequently challenge these plants with TYLCV. Our data demonstrate that the CRISPR/Cas9 system targeted TYLCV for degradation and introduced mutations at the target sequences. All tested sgRNAs exhibit interference activity, but those targeting the stem-loop sequence within the TYLCV origin of replication in the intergenic region (IR) are the most effective. N. benthamiana plants expressing CRISPR/Cas9 exhibit delayed or reduced accumulation of viral DNA, abolishing or significantly attenuating symptoms of infection. Moreover, this system could simultaneously target multiple DNA viruses.These data establish the efficacy of the CRISPR/Cas9 system for viral interference in plants, thereby extending the utility of this technology and opening the possibility of producing plants resistant to multiple viral infections.