Center for Genome Engineering, Institute for Basic Science, Daejeon, Republic of Korea.
Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Nat Commun. 2022 Jul 12;13(1):4038. doi: 10.1038/s41467-022-31745-y.
Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddA, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size. Here, we present non-toxic, full-length DddA variants to make monomeric DdCBEs (mDdCBEs), enabling mitochondrial DNA editing with high efficiencies of up to 50%, when transiently expressed in human cells. We demonstrate that mDdCBEs expressed via AAV in cultured human cells can achieve nearly homoplasmic C-to-T editing in mitochondrial DNA. Interestingly, mDdCBEs often produce mutation patterns different from those obtained with conventional dimeric DdCBEs. Furthermore, mDdCBEs allow base editing at sites for which only one TALE protein can be designed. We also show that transfection of mDdCBE-encoding mRNA, rather than plasmid, can reduce off-target editing in human mitochondrial DNA.
细菌间毒素 DddA 衍生的胞嘧啶碱基编辑器（DdCBE）可实现核和细胞器 DNA 中的靶向 C 到 T 转换。DddA 是源自伯克霍尔德氏菌的脱氨酶催化结构域，当其与转录激活样效应物（TALE）DNA 结合蛋白融合以制造 DdCBE 时，为避免其在真核细胞中的细胞毒性，被分割成两个无活性的半体。因此，DdCBE 作为一对起作用，这阻碍了通过小载体大小的病毒载体进行基因传递。在这里，我们提出了非毒性的全长 DddA 变体，以制造单体 DdCBE（mDdCBE），当在人细胞中转瞬表达时，可实现高达 50%的高效线粒体 DNA 编辑。我们证明，通过 AAV 在培养的人细胞中表达的 mDdCBE 可以实现线粒体 DNA 中近乎同质的 C 到 T 编辑。有趣的是，mDdCBE 通常产生不同于传统二聚体 DdCBE 获得的突变模式。此外，mDdCBE 允许针对只能设计一个 TALE 蛋白的位点进行碱基编辑。我们还表明，mDdCBE 编码 mRNA 的转染，而不是质粒，可以减少人线粒体 DNA 中的脱靶编辑。
