Plant viral suppressors HcPro and P19 inhibit CRISPR-Cas13-mediated transcript interference

Abstract RNA plays a central role in cellular dynamics, mediating the formation of proteins from genetic information. Thus, there is great interest in technologies that can alter RNA content of a cell to achieve desired outcomes. RNA targeting CRISPR‒Cas13 enables precise, sequence-guided targeting of RNA to disrupt translation and can therefore be used for cellular engineering. However, guide RNA from CRISPR systems can silence target transcripts independent of the Cas13 protein, complicating the deployment of CRISPR-Cas13 for biotechnology. To prevent Cas13-independent silencing and encourage Cas13-mediated transcript reduction, we explored the use of viral suppressors of RNAi. Here, we report that expression of two viral suppressors of RNAi, HcPro and P19, unexpectedly blocked Cas13-mediated transcript silencing in addition to Cas13-independent RNAi-like silencing. To further understand how HcPro blocks Cas13-mediated silencing, we expressed an RNA silencing-deficient HcPro mutant for RNA-targeting assays. The results demonstrate that mutant HcPro failed to suppress Cas13-dependent silencing, indicating that HcPro RNAi suppression also interferes with Cas13 silencing. These results suggest that plant viruses may be primed to overcome engineered CRISPR-Cas13 antiviral resistance through the activity of previously evolved RNAi silencing suppressors.