Sequence‐informed, Cas13‐based Technologies for RNA Viruses

Abstract

Infectious diseases, many caused by RNA viruses, remain a significant global health threat. Three recent events — the Ebola virus epidemic from 2013–2016, the Zika virus pandemic from 2015–2016, and the ongoing severe acute respiratory syndrome coronavirus 2 pandemic — have illustrated that our ability to diagnose and treat known or newly emerging viral threats is severely limited, largely due to RNA viruses’ high diversity and rapid evolution. However, the highly programmable nature of CRISPR-Cas systems as well as continued discovery of new DNA- and RNA-targeting Cas proteins can accelerate the development of alternative diagnostic and therapeutic approaches. In this dissertation, I demonstrate how growing viral genomic information along with the discovery and use of an RNA-guided, RNA-targeting CRISPR effector protein, Cas13, have enabled the development of technologies that can address unmet diagnostic and therapeutic needs. In Chapter 2, I applied multiplexed amplification methods and next-generation sequencing to generate Zika virus genomic data from patient samples collected during the 2015–2016 pandemic in the Americas. Further, I describe both diagnostic and sequencing challenges associated with low-titer viral infections that this pandemic illuminated. These challenges subsequently motivated me to develop the technologies I describe in the following chapters. In Chapter 3, I developed Cas13-based viral diagnostics that can be used in low-resource settings. These diagnostics are specific, sensitive, user-friendly, and have limited equipment requirements. Because of these advancements, I deployed Cas13-based diagnostics for Zika and dengue viruses in Honduras. In Chapter 4, I harnessed Cas13’s sequence-guided, RNase activity to target and inhibit multiple RNA viruses in mammalian cell culture. I systematically tested hundreds of virus-specific Cas13 CRISPR-RNAs to characterize principles governing Cas13 targeting of viral RNA, and I highlighted the unique capacity of Cas13 to act as an end-to-end system for both detection and destruction of RNA viruses. In addition, I used next-generation sequencing to characterize differences in both the host transcriptome and viral population due to Cas13 targeting. In summary, this dissertation demonstrates the combined power of viral genomics and the RNA-guided, RNA-targeting activity of Cas13 to transform diagnosis and treatment of infectious diseases.