Viral Genome RNA Destruction Using the CRISPR/Cas Effector C2C2

Viral infections account for major economic losses in livestock and are major threats to the public health. Most of existing approaches to control viral infections are based on prophylaxes (immunization); however, there are limited options after the onset of an infection. Resistance against New Castle disease virus and Avian Influenza through engineered resistance by genomic editing could be a novel way to combat these viral diseases. The proposed project is a novel strategy to restrict the ongoing virus replications by specifically targeting the viral RNA using a modified Crispr/CAS9 system.
This studentship will apply a newly discovered RNA-guided RNAase function of Class 2 Type VI-A effector C2C2 to specifically block viral replication by cleaving viral RNA. We propose to streamline the approach and to provide a proof-of-principle concept as a novel antiviral approach. Based on C2c2-mediated RNA cleavability in prokaryotes, our experience with genome editing, and our preliminary work, the approach is viable, effective and may open future avenues to study viral and host RNA metabolism in vitro and in vivo.
Genome editing mediated by clustered regularly interspaced short palindromic repeats (CRISPR) and nucleases (e.g. Cas9) technology has revolutionized the genetic modifications of endogenous genes in a wide variety of biomedically important cell types and organisms (Cong et al., 2013). However, all functionally characterized CRISPR/Cas systems have been reported to target DNA. These systems are thus ideal to manipulate DNA in both hosts and viruses (e.g. DNA viruses such as Herpesviruses). Given that no DNA step is involved in the replication of RNA viruses, CRISPR/Cas9 system can't be conveniently applied to study the biology of RNA viruses. The only RNA-targeted system, available so far, was reported by Feng Zhang (pioneer of CRISRP/Cas9 at Massachusetts Institute of Technology, USA) (Abudayyeh et al., 2017 Science). While this bacterial adaptive system appeared ideal to target RNA in prokaryotes, the C2c2 system can be exploited as an antiviral approach. Owing to high RNAse activities of HEPN domain of C2c2, this RNA-guided RNA-targeting CRISPR effector is a feasible approach to cleave viral RNA and to cripple virus replication. At this stage, we will apply this approach against Newcastle disease virus and influenza virus, which are single stranded RNA viruses of poultry. Our experience with genome editing at The Roslin Institute and the capacity to work with pathogenic viruses at the Roslin Institute will allow the student to investigate the potential of C2c2 in blocking RNA virus replication. This investigation would provide tools which may someday produce livestock with resistance to the major viral pathogens.