Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddA tox -derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at T C or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for G C targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddA tox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddA tox , which catalyzed C-to-T editing at both HC and G C targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddA tox - or riDddA tox -mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddA tox -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-T C targets. [Display omitted] • A peculiar dsDNA deaminase from Roseburia intestinalis is identified • riDddA tox -composed CBEs facilitate DNA editing without sequence constraint • Transactivator fusion enhances editing capability of crDdCBEs and mitoCBEs • riDddA tox -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. [ABSTRACT FROM AUTHOR]