Gupta Darshana, Patinios Constantinos, Bassett Harris V, Kibe Anuja, Collins Scott P, Kamm Charlotte, Wang Yanyan, Zhao Chengsong, Vollen Katie, Toussaint Christophe, Calvin Irene, Cullot Grégoire, Aird Eric J, Polkoff Kathryn M, Nguyen-Vo Thuan Phu, Migur Angela, Schut Friso, Al'Abri Ibrahim S, Achmedov Tatjana, Del Re Alessandro, Corn Jacob E, Saliba Antoine-Emmanuel, Crook Nathan, Stepanova Anna N, Alonso Jose M, Beisel Chase L
Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz Centre for Infection Research (HZI), Würzburg, Germany.
Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.
Nat Biotechnol. 2025 Sep 4. doi: 10.1038/s41587-025-02802-w.
Base editors create precise genomic edits by directing nucleobase deamination or removal without inducing double-stranded DNA breaks. However, a vast chemical space of other DNA modifications remains to be explored for genome editing. Here we harness the bacterial antiphage toxin DarT2 to append ADP-ribosyl moieties to DNA, unlocking distinct editing outcomes in bacteria versus eukaryotes. Fusing an attenuated DarT2 to a Cas9 nickase, we program site-specific ADP-ribosylation of thymines within a target DNA sequence. In tested bacteria, targeting drives homologous recombination, offering flexible and scar-free genome editing without base replacement or counterselection. In tested yeast, plant and human cells, targeting drives substitution of the modified thymine to adenine or a mixture of adenine and cytosine with limited insertions or deletions, offering edits inaccessible to current base editors. Altogether, our approach, called append editing, leverages the addition of chemical moieties to DNA to expand current modalities for precision gene editing.
碱基编辑器通过引导核碱基脱氨或去除来创建精确的基因组编辑，而不会诱导双链DNA断裂。然而，用于基因组编辑的其他DNA修饰的巨大化学空间仍有待探索。在这里，我们利用细菌抗噬菌体毒素DarT2将ADP-核糖基部分附加到DNA上，在细菌和真核生物中解锁不同的编辑结果。将减毒的DarT2与Cas9切口酶融合，我们对目标DNA序列中的胸腺嘧啶进行位点特异性ADP-核糖基化编程。在测试的细菌中，靶向驱动同源重组，提供灵活且无疤痕的基因组编辑，无需碱基替换或反选择。在测试的酵母、植物和人类细胞中，靶向驱动修饰的胸腺嘧啶替换为腺嘌呤或腺嘌呤和胞嘧啶的混合物，并伴有有限的插入或缺失，提供了当前碱基编辑器无法实现的编辑。总之，我们的方法称为附加编辑，利用向DNA添加化学部分来扩展当前的精确基因编辑模式。
