Examination of the Cell Cycle Dependence of Cytosine and Adenine Base Editors

Abstract

Base editors (BEs) are genome editing agents that install point mutations with high efficiency and specificity. Due to their reliance on uracil and inosine DNA damage intermediates (rather than double-strand DNA breaks, or DSBs), it has been hypothesized that BEs rely on more ubiquitous DNA repair pathways than DSB-reliant genome editing methods, which require processes that are only active during certain phases of the cell cycle. We report here the first systematic study of the cell cycle-dependence of base editing using cell synchronization experiments. We find that nickase-derived BEs (which introduce DNA backbone nicks opposite the uracil or inosine base) function independently of the cell cycle, while non-nicking BEs are highly dependent on S-phase (DNA synthesis phase). We found that synchronization in G1 (growth phase) during the process of 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 observe that endogenous expression levels of DNA damage repair pathways are sufficient to process base editing intermediates into desired editing outcomes, and the process of base editing does not significantly perturb transcription levels. Overall, our study provides mechanistic data demonstrating the robustness of nickase-derived BEs for performing genome editing across the cell cycle.

Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors

“…Changing to n’Cas9 results in an even higher degree of TS mutations alone, although the fact that the nick is positioned downstream of the mutations likely limits the ability of DNA replication to install mutations as readily. These results highlight the importance of downstream DNA repair pathways in either resolving or embedding mutations, aligning with recent work that highlights the cell cycle dependence of base editing outcomes ( 47 ).…”

Cooperativity between Cas9 and hyperactive AID establishes broad and diversifying mutational footprints in base editors

“…Changing to n’Cas9 results in an even higher degree of TS mutations alone, although the fact that the nick is positioned downstream of the mutations likely limits the ability of DNA replication to install mutations as readily. These results highlight the importance of downstream DNA repair pathways in either resolving or embedding mutations, aligning with recent work that highlights the cell cycle dependence of base editing outcomes (46).…”

Epitope base editing CD45 in hematopoietic cells enables universal blood cancer immune therapy

“…Various gene-editing technologies can be used to edit the targeted epitope in such a way that it prevents CAR45 recognition. We chose CRISPR base editing over nuclease-based homology-directed repair (HDR) knock-in because base editing is largely cell cycle independent ( 38 ), results in lower indel formation, has fewer off-target editing events, and yields higher purity (a high percentage of all edited alleles that contain the requisite base conversion without indels) without causing double-strand breaks ( 39 ). This mitigates the risk of chromosomal translocations, potentially toxic DNA damage responses, and activation of innate immune receptors sensing the DNA donor template ( 40 , 41 ).…”