UC San Diego

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.