A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility

• Published in: Molecular cell 2023-05, Vol.83 (10), p.1710-1724.e7
• Main Authors: Guo, Junfan, Yu, Wenxia, Li, Min, Chen, Hongyu, Liu, Jie, Xue, Xiaowen, Lin, Jianxiang, Huang, Shisheng, Shu, Wenjie, Huang, Xingxu, Liu, Zhen, Wang, Shengqi, Qiao, Yunbo
• Format: Article
• Language: English
• Subjects: Animals; CRISPR-Cas Systems; Cytosine; DdCBE; DNA, Mitochondrial - genetics; Gene Editing; Mice; mitoCBE; mitochondrial DNA; mtDNA; Mutation; non-TC; nuclear DNA; Roseburia intestinalis; Trans-Activators - metabolism; transactivator
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2023.04.012

Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets. [Display omitted] •A peculiar dsDNA deaminase from Roseburia intestinalis is identified•riDddAtox-composed CBEs facilitate DNA editing without sequence constraint•Transactivator fusion enhances editing capability of crDdCBEs and mitoCBEs•riDddAtox-mediated mitoCBEs stimulate disease-associated mtDNA mutations in vivo Guo et al. engineered nuclear and mitochondrial DNA base editors based on a peculiar double-stranded cytosine deaminase, which can efficiently catalyze C-to-T conversions without sequence constraint assisted by transcriptional activator fusion. It provides a useful tool with expanded target compatibility and enhanced activity.