DNMT3A（DNA 甲基转移酶3A）是一个新型胞嘧啶甲基转移酶，负责在哺乳动物发育过程中建立适当的DNA甲基化。DNMT3A的缺失功能（LOF）突变，包括热点突变 R882H，在发育性生长障碍以及血液病，包括克隆性造血和急性髓细胞白血病中经常发生。因此，识别激活DNMT3A的机制对于基础研究和治疗有重要意义。在这里，我们应用了一个基因编辑器突变扫描策略，结合改进的DNA甲基化报告基因，系统地在细胞中鉴定DNMT3A激活突变。通过将优化的细胞招募策略与具有或不具有LOF热点突变 R882H 的成对同源细胞系整合，我们鉴定并验证了在DNMT3A的调节 ADD 结构域内或相互作用的三个不同的超活化突变，将这些区域提名为可能的药物干预的功能靶点位。值得注意的是，这些突变在异源 R882H 突变的背景下仍然具有激活功能。总之，我们展示了基因编辑器扫描在发现靶蛋白功能区域方面的实用性。

DNA methyltransferase 3A (DNMT3A) is a de novo cytosine methyltransferase responsible for establishing proper DNA methylation during mammalian development. Loss-of-function (LOF) mutations to DNMT3A, including the hotspot mutation R882H, frequently occur in developmental growth disorders and hematological diseases, including clonal hematopoiesis and acute myeloid leukemia. Accordingly, identifying mechanisms that activate DNMT3A is of both fundamental and therapeutic interest. Here, we applied a base editor mutational scanning strategy with an improved DNA methylation reporter to systematically identify DNMT3A activating mutations in cells. By integrating an optimized cellular recruitment strategy with paired isogenic cell lines with or without the LOF hotspot R882H mutation, we identify and validate three distinct hyperactivating mutations within or interacting with the regulatory ADD domain of DNMT3A, nominating these regions as potential functional target sites for pharmacological intervention. Notably, these mutations are still activating in the context of a heterozygous R882H mutation. Altogether, we showcase the utility of base editor scanning for discovering functional regions of target proteins.