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Mitochondrial base editor induces substantial nuclear off-target mutations

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DddA-derived cytosine base editors (DdCBEs), which are fusions of the split-DddA halves and transcription activator-like effector (TALE) array proteins, enable targeted C·G-to- T·A conversions in mitochondrial DNA1. However, its genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or -independent. TAS-dependent off-target sites in the nuclear DNA (nDNA) are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by a paired TALE proteins positioned in close proximity. TAS-independent nDNA off-target sites are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with CTCF-binding sites and are enriched in TAD boundaries. We also engineered DdCBE to alleviate such off-target effect. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base editing tools.

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Correspondence to Chengqi Yi.

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This file contains Supplementary Discussion; legends for Supplementary Tables 1-5 and Supplementary Videos 1-2; Supplementary Figures 1-27 and Supplementary References.

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Lei, Z., Meng, H., Liu, L. et al. Mitochondrial base editor induces substantial nuclear off-target mutations. Nature (2022).

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