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

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.

本研究首次通过细胞同步化实验，系统探究了碱基编辑（base editing）的细胞周期依赖性。我们发现，切口酶衍生碱基编辑器（Base Editors，BEs）可在尿嘧啶或肌苷碱基的互补链引入DNA主链切口，其功能不依赖于细胞周期；而非切口型碱基编辑器则高度依赖S期（DNA合成期，S-phase）。我们还发现，在胞嘧啶碱基编辑过程中对细胞进行G1期（生长阶段）同步化，会显著提升C:G到A:T副产物的引入率，这一发现可用于开发精准C:G到A:T碱基编辑的新策略。此外，我们观察到DNA损伤修复通路的内源性表达水平足以将碱基编辑中间体加工为预期的编辑产物，且碱基编辑过程不会显著干扰转录水平。