The Transposon-Encoded Protein TnpB is an RNA-Guided Nuclease

Abstract

Programmable RNA-guided systems, such as CRISPR-Cas adaptive immune systems, perform a wide variety of biological functions and have been harnessed for important therapeutic and diagnostic biotechnologies. One CRISPR effector protein, the Cas12 nuclease, is hypothesized to have its evolutionary origins in TnpB, an abundant transposon-encoded protein that contains a predicted nuclease domain. The biochemical properties of TnpB are unknown, but may provide clues to the evolutionary origins of RNA-guided activity.

In this thesis, we investigate the biochemical activity of TnpB, revealing that this protein uses a noncoding RNA as a guide to cleave DNA substrates in a targeted manner. We investigate the biogenesis of the guide RNA, finding that TnpB can process its own mRNA to yield the guide RNA. In the process of studying guide RNA biogenesis, we also identify a potential cis-regulatory mechanism whereby part of the TnpB mRNA downregulates DNA cleavage activity. Next, we study the biochemical diversity of this protein across 59 diverse TnpB orthologs, and identify conserved features of TnpB function. Finally, we evaluate the prospects for TnpB as a human genome editing tool and highlight structural features that govern activity and specificity in human cells.

Ultimately, TnpB is emblematic of a new class of programmable RNA-guided systems, the Obligate Mobile Element Guided Activity (OMEGA) family. This work serves as a window into the diversity of RNA-guided functions in nature and provides a rich source for future biotechnological development.