Palindromic sequence zinc finger12/22/2023 Mutant mtDNA coexists with wild-type mtDNA, and the ratio of wild-type to mutant mtDNA often correlates with the severity of the clinical phenotype 4. Most human cells with mitochondrial disease have heteroplasmic mtDNA that exists in multiple copies. Although the CRISPR system has been widely used for nuclear genome base editing 1, 2, it is currently impractical to apply this system for editing the mitochondrial genome due to the absence of an effective method for delivering guide RNA into this organelle 3. Therefore, there is a high demand for technologies that enable mtDNA base editing, which could aid in understanding the underlying mechanisms of pathogenesis and developing cures for these diseases. Mitochondrial DNA (mtDNA) mutations are associated with many human diseases, and around 95% of these are point mutations that could potentially be corrected using base editing approaches. mitoBEs offer a precise, efficient DNA editing tool with broad applicability for therapy in mitochondrial genetic diseases. Furthermore, we correct pathogenic mitochondrial DNA mutations in patient-derived cells by delivering mitoBEs encoded in circular RNAs. We find that mitoBEs are DNA strand-selective mitochondrial base editors, with editing results more likely to be retained on the nonnicked DNA strand. Combining mitochondria-localized, programmable TALE binding proteins with the nickase MutH or Nt.BspD6I(C) and either the single-stranded DNA-specific adenine deaminase TadA8e or the cytosine deaminase ABOBEC1 and UGI, we achieve A-to-G or C-to-T base editing with up to 77% efficiency and high specificity. In this study, we present mitochondrial DNA base editors (mitoBEs), which combine a transcription activator-like effector (TALE)-fused nickase and a deaminase for precise base editing in mitochondrial DNA. A number of mitochondrial diseases in humans are caused by point mutations that could be corrected by base editors, but delivery of CRISPR guide RNAs into the mitochondria is difficult.
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