Discovery and characterization of diverse microbial RNA-guided systems

Abstract

Precise modification of nucleic acids is a powerful technique to enable understanding of the relationship between variation in genetic information and biological phenotypes and to treat genetic diseases. Over the past decade, the microbial adaptive immune system CRISPR-Cas has revolutionized genome editing, largely due to ease of target reprogramming. Cas nucleases can be retargeted by changing a short sequence in the associated non-coding RNA, which acts to guide the enzyme or complex to a complementary nucleic acid target. Exploration of the natural diversity of CRISPR-Cas systems has uncovered numerous variants with widely differing protein and domain architectures that exhibit distinct substrate specificities and activities, tying RNA-guided nucleic acid recognition to diverse enzymatic functions. This diversity has fueled the development of CRISPR-Cas systems for additional applications beyond genome editing, such as transcriptome editing, rapid diagnostics and molecular recording tools, and has aided in optimization of existing technologies via identification of systems that naturally exhibit desirable properties.

In this thesis, we explore the evolution and diversity of RNA-guided microbial systems and characterize and engineer them for use in human cells. First, we investigate the origins of Cas9 and Cas12, the most widely used Cas proteins for genome editing. We find that they evolved from compact transposon-encoded nucleases, which we termed OMEGA, that already employed an RNA-guiding mechanism. Next, we develop OMEGA systems as genome editing tools that, due to their small size, are more compatible with therapeutic delivery vectors. We further explore microbial genomes and metagenomes to discover Cas13b-t, a similarly compact RNA-targeting system that we develop as a deliverable RNA editing platform. Finally, we extended the theme of mining sequence databases to comprehensively catalog proteins and domains associated with CRISPR-Cas systems. Through this analysis, we identify and characterize several novel CRISPR-Cas types and subtypes with potential for development as biotechnologies.