UC San Diego

Base editors are tools that chemically modify the nucleobases of DNA and RNA in a programmable manner, allowing for genome, epigenome, and transcriptome editing in live cells. These tools can be used to introduce specific base transitions in DNA or RNA, manipulate methylation patterns in the epigenome, and create genetically encoded libraries in target genes. These various functions can be used to modulate every aspect of the central dogma. The efficiency and precision of base editors makes them useful in both basic research and in the development of new therapies. The adenosine base editor (ABE) was developed by evolving the RNA-modifying enzyme TadA to also accept DNA as substrate. This suggests that the base editing tool kit can be expanded to use many of the naturally occurring RNA-modifications as intermediates. However, current base editors remain limited to deaminases, suggesting that more complex chemical reactions face additional barriers. To better inform the creation of new base editors, we set out to better understand the molecular mechanisms of the wild-type TadA enzyme (used in ABE0.1) and the DNA-modifying variants developed for base editing (ABE7.10, ABE8e, ABE8.20, etc.). First, we observe that ABE0.1 shows high Cas9-independent, off-target RNA-editing at UACG motifs in accessible loops. In chapter 3, we ask if this discovery also extends to DNA and find that ABE0.1 can perform targeted DNA-editing at TACG motifs. This editing is inefficient though and may be easily missed, so we leveraged this discovery to develop a fluorescent reporter of base editing that greatly increases our ability to detect low-levels of base editing. We finish out the chapter by using this fluorescent reporter to better understand the sequence-dependent editing of ABE0.1 and evolved ABEs. Finally, we apply this new understanding of an early base editor to develop more sequence-optimized fluorescent reporters to: detect DNA editing by RNA-modifying enzymes that could be used for base editing in chapters 4 and 5, develop a protocol for evolving new base editors in mammalian cells in chapter 6, and developing an outreach experiment to introduce base editing to high school students in chapter 7. Through the experiments in each of these sections, we gain a better understanding of how the sequence recognition properties of RNA-modifying enzymes can be leveraged to develop new base editors.