Precision mitochondrial genome editing with unconstrained DdCBEs

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C-to-T conversions in mitochondrial DNA (mtDNA). DdCBEs work in pairs, with each arm composed of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5'-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this requirement, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5'-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs containing TALE proteins engineered to recognize all 5' bases. These modified DdCBEs are herein referred to as unconstrained DdCBEs. Notably, 5'-T-noncompliant canonical DdCBEs efficiently edited mtDNA at diverse loci. However, they were frequently outperformed by unconstrained DdCBEs, which exhibited significant improvements in activity and specificity, regardless of the 5'-most bases of their TALE binding sites. Furthermore, we showed that unconstrained DdCBEs are compatible with the enhanced DddAtox variants DddA6 and DddA11, and we validated TALE shifting with unconstrained DdCBEs as an effective approach to optimize base editing outcomes. Overall, unconstrained DdCBEs enable efficient, specific, and sequence-unrestricted mitochondrial base editing.