![]() Because TadA is an essential tRNA-specific adenosine deaminase originating from Escherichia coli 15, it is unsurprising that TadA8e-V106W enables mtDNA editing. We detected a very low level of editing at all three targeting sites, MT-ND1, MT-ND4 and MT-RNR2, with an editing rate of up to 0.39%, which is barely above the deep sequencing error (>0.10%) (Fig. To investigate the potential for mitochondrial A-to-G editing, we fused the TadA8e-V106W protein with the appropriate TALE array and mitochondrial targeting sequence. TadA8e-V106W is an engineered deoxyadenosine deaminase that is commonly fused with Cas protein to perform adenosine-to-inosine (recognized as guanine) editing on nuclear DNA 14. Insufficiency of TALE–TadA8e-V106W alone to facilitate the editing of mtDNA To this end, we developed a method for efficient and accurate mtDNA base editing by combining a deaminase and nickase. Given that deamination of either C or A should not occur while in the state of base pairing 1, we hypothesized that introducing single-stranded DNA (ssDNA) around the target loci could enable targeted deamination of mtDNA. In addition, DddA’s direct or indirect interaction with CTCF can result in a broad range of off-target effects on the nuclear genome 13. However, single TALE binding can lead to off-target effects in the split DddA halves. DdCBEs involve the fusion of split DddA halves, TALE array proteins and uracil glycosylase inhibitor (UGI) 9, 10, 11, whereas TALEDs combine TALE, DddA and deaminase to achieve A-to-G editing in mitochondria 12.īoth DdCBEs and TALEDs perform base deamination on both strands of double-stranded DNA (dsDNA) within the editing window. Recently, DdCBEs (DddA-derived cytosine base editors) 9, 10, 11 and TALEDs (transcription-activator-like effector-linked deaminases) 12 have been developed to achieve C-to-T and A-to-G conversions, respectively, in mtDNA. ![]() However, these approaches are not suitable for treating mitochondrial diseases involving homogeneous mutations and do not support the introduction of new sequence changes. Researchers have fused mitochondrial targeting sequences with RNA-free programmable nucleases, such as zinc-finger nucleases and transcription activator-like effector (TALE) nucleases (TALENs), to achieve targeted degradation of mutant mtDNA and increase the proportion of wild-type mtDNA 5, 6, 7, 8. 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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