CRISPR-RfxCas13d-Mediated Antiviral Therapeutic Against Venezuelan Equine Encephalitis Virus RNA-Dependent RNA-Polymerase

Venezuelan Equine Encephalitis Virus (VEEV) is an emerging enveloped, arthropod-transmitted, single-stranded, and positive-sense RNA virus. VEEV is part of the Togaviridae Family genus of Alphaviruses, responsible for highly pathogenic mosquito-borne viruses that cause 90% mortality rates in equine animals, including horses, donkeys, zebras, and mules, while inducing 1% mortality in humans with 14% of infected persons suffering severe neurological diseases. VEEV has historically been engineered by the USA and the former USSR during the Cold War as a biological agent to target developing, agrarian-oriented countries that primarily rely on equines for industrial and agricultural production. Since VEEV infects the central nervous system, is highly pathogenic, and can be aerosolised, the CDC and NIAID considers it a Biosafety Level 3 pathogen and a Category B weapon of biowarfare and bioterrorism. Few FDA-approved medical countermeasures exist for VEEV and other Alphaviruses. The TC-83 vaccine strain is employed for military and agricultural personnel and horses but is not USA FDA-approved. Our research aims to develop an antiviral therapeutic using Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated Systems (CRISPR-Cas), particularly RfxCas13d which is a bacterial antiviral endonuclease. We employ Cas13design computationally derived Guide-RNA that will complementarily bind to VEEV genomic RNA-dependent RNA-polymerase (RdRp) and prime it for RNA degradation by RfxCas13d. VEEV-eGFP Replicon systems are employed to express RdRp in cell culture, with fluorescence intensities reported to measure RNA degradation. Further live virus testing will validate VEEV-specific RfxCas13d-gRNAs as a potential antiviral strategy against VEEV replication and transcription.