Immunostimulatory guide RNAs mediate potent antiviral response

Genome-editing with CRISPR has emerged as a technology with broad therapeutic potential. However, it is unclear whether CRISPR will elicit innate immune responses, which could impact both positively and negatively on the desired therapeutic effects. Here, we have examined the immune-stimulatory properties of different variants of guide RNAs (gRNAs) – in vitro transcribed gRNA (IVT-gRNA) and synthetic gRNAs with or without chemical modifications, full-length or duplexed. We find that only IVT-gRNA evokes strong expression of cytokines in a panel of cell lines while all the synthetic RNAs do not. We further find that sensing of IVT-gRNA proceeds mainly through the RIG-I/MAVS RNA sensing axis. One potential use of CRISPR is for antiviral therapy. The antiviral actions of the gRNA tested up until now have been relying purely on the gene editing function of the CRISPR machinery, which weakens its feasibility due to the difficulty to target all infected cells. When IVT-gRNA was combined with unmodified Cas9 mRNA, which also induces cytokine expression, strong immune response was obtained while maintaining nuclease activity of CRISPR. Remarkably, such combination inhibited herpes simplex virus type-1 (HSV-1) replication even though the nuclease activity was modest, and provided ‘bystander protection’ to the cells that were not transfected with CRIPSR molecules. The antiviral activity of IVT-gRNA was also observed in vivo in HSV-1-infected Cas9+ mice, thus demonstrating the therapeutic potential. Our study further extends the applications of CRISPR by exploiting the immunostimulatory function of gRNAs.