UC Berkeley

The natural world encompasses an awesome diversity of organisms, each which have evolved molecules and molecular assemblies enabling them to survive. From an engineering perspective, these molecular tools compose a huge reservoir of evolutionarily-honed building blocks that can be harnessed to create solutions to emerging problems. In this dissertation, I describe two projects that leverage modules derived from bacteria and viruses to address problems in human health.

One strategy that has quickly been adopted and manipulated by the scientific community is the CRISPR-Cas system. CRISPR-Cas systems evolved in prokaryotes as an adaptive immune response against ever-evolving viruses. These systems function by complexing effector protein(s) with RNA that targets the complex to invasive genetic material for degradation. The effector proteins from these systems, namely Cas9, 12, and 13 have been repurposed for many uses – from therapeutic gene editing to molecular diagnostics. Cas13a is an RNA-guided RNAse that targets specific RNA sequences and, once activated by its target, non-specifically cleaves other RNA segments. CRISPR-Cas13a has been repurposed as a tool for molecular diagnostics of SARS-CoV-2 and other viral RNA in patient samples. We sought to extend this strategy to detect small non-coding RNAs which play a key role in regulating cells. Specifically, we wanted to detect miRNAs (~17-26nt long) and smaller RNAs, which have emerged as promising biomarkers for disease diagnosis. In this work, I demonstrate that Cas13a can detect RNA segments as small as eight nucleotides by splitting the guide RNA. Critically, we show that the Cas13a split guide system can detect miRNAs in cell lysate – without the need to purify or amplify the target. This project introduces a new Cas-based tool for small RNA diagnostics and biology studies.

Another reservoir of biological building blocks is the world of viruses. Viruses have evolved to be excellent at entering hosts, infiltrating their cells, and commandeering the cell to make more virions. Consider Influenza A virus (IAV): an endemic virus and that has caused at least six major pandemics. Here, I present a strategy to turn that pathogen into a partner, leveraging its unique genetic and biophysical properties to create a delivery vehicle to transport gene editing tools to hard-to-reach mucous-protected cells. I focus on delivering CRISPR-Cas9 as a ribonucleoprotein (RNP) instead of as a gene cassette, addressing issues of inefficient and off-target gene editing. To achieve this goal, I trick the native IAV genome packaging machinery to incorporate Cas9 guide RNA disguised as viral RNA into a non-replicating Influenza A virus-like particle. The Cas9 protein binds to the chimeric guide RNA, piggybacking into the budding particle to form a Cas9 Influenza-based Delivery Vehicle (CasIDV). We show preliminary evidence that CasIDVs deliver Cas9 RNPs to cultured cells, resulting in gene editing events. With further optimization, the CasIDV system has the potential to become a promising strategy to deliver therapeutic cargo like CRISPR-Cas gene editing tools to currently inaccessible cells.