Characterizing and Reducing Spurious DNA Edits by CRISPR Cytosine Base Editors

Abstract

Cytosine Base Editors (CBEs) enable precise cytosine-to-thymine genetic mutations via APOBEC-mediated deamination of CRISPR-targeted cytosines. However, due to the natural RNA- and DNA-editing capacity of the APOBEC domains they harbor, CBEs possess the ability to create substantial pseudorandom cytosine editing events across the genomes and transcriptomes of affected cells in a gRNA-independent manner. These events, here termed spurious deamination or spurious editing, may represent a major barrier toward adapting CBEs to clinical use, since they create unpredictable and potentially deleterious effects on the cells they inhabit. Here, I describe the development of a class of CBEs made from split deaminase domains that possess a reduced capacity for spurious editing than monomeric CBEs, possibly by increasing the molecularity of spurious editing events and thereby decreasing their associated reaction rates. I characterize the utility of this novel class of CBEs,and develop a facile in situ experimental method called Base Editing at Anchored R-Loop DNA (BE-ARD) to assess their capacity for carrying out spurious DNA edits. Chapter 1 describes the relevant background of the CRISPR base editor field, focusing on Cytosine-to-Thymine base editors (CBEs), as well as my efforts to characterize and minimize all dimensions of deleterious effects using split-deaminase base editors, and its discussion includes a description of a machine-learning algorithm to investigate the biochemical parameters governing observed editing outcomes. Chapter 2 describes an attempted genome-scale CRISPR screen using targeted integrations to follow the distributions of edits throughout a population of cells.