The development of CRISPR-derived genome editing technologies enables precise manipulation of DNA sequences within the human genome. One class of these editing tools is base editors, which install point mutations with high efficiency and specificity, without the disadvantages of traditional Cas9-based nucleases. This is due to the introduction of uracil and inosine intermediates, as opposed to breakage of the DNA backbone. As such, they rely on more ubiquitous DNA repair pathways than wild type Cas9. Cas9 is also highly reliant on cell cycle phase, depending on homology-directed repair (HDR), which is only active in late S and G2 phases. To date, no one has investigated the relationship of base editors to the cell cycle. We examine how reliant base editors are on the cell cycle using chemical synchronization experiments. We optimize protocols to evaluate base editing under synchronization conditions. We find that nickase-derived BEs function independently of the cell cycle, while non-nicking variants are highly dependent on S-phase processes. We discover that G1 synchronization during cytosine base editing causes significant increases in C•G to A•T “byproduct” introduction rates, which can be leveraged to discover new strategies for precise C•G to A•T base editing. We investigate the cause for C•G to A•T editing events and design an integrated fluorescent reporter system to identify genes implicated in these editing outcomes. We discover TLS polymerases POLI and POLK are likely responsible for these unique editing events. We further examine base editors’ response to nucleotide concentration to identify if that alters editing outcome. Finally, we develop and test a cell-cycle regulated base editor to apply our discovery towards technology development.

Base editing is a recent development in the genome editing field that allows for the introduction of single nucleotide changes in the genome with high efficiency. This is accomplished without the induction of double-strand breaks (DSBs), a hallmark of traditional Cas9 editing that suffers from modest editing efficiencies, and high frequencies of random insertions and deletions (indels) instead of the desired genome editing product. Base editing technology in its current form still suffers from shortcomings, such as undesired genome editing byproducts, that demand a more thorough understanding of its cellular mechanisms. A major limitation of DSB-reliant genome editing methods is that their activity fluctuates in different phases of the cell cycle. To initiate our investigations of the cellular mechanism of base editing, we chemically synchronized cells into the various stages of the cell cycle and measure the effects this had base editing efficiencies and levels of byproduct formation. We observed a decrease in base editing byproduct formation when cells were synchronized in G1, the cell cycle phase when cells are growing but not replicating their DNA. To further characterize base editing mechanisms we have developed a genome-wide screen to identify the key proteins involved in repair of uracil-guanine mismatches, the intermediates of cytosine base editing.