Mitochondria, the “powerhouses” of cells, play a vital role in adenosine triphosphate (ATP) production. Mitochondria possess their own independent DNA, termed mitochondrial DNA (mtDNA), which encodes genes essential for mitochondrial function. Compared to nuclear DNA, mtDNA is more prone to mutations due to reactive oxygen species generated during oxidative phosphorylation. Most mtDNA mutations are base substitutions, leading to mitochondrial genetic disorders such as Leber hereditary optic neuropathy (LHON), Leigh syndrome, and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS).
Among known disease-associated base substitutions, over 40% are G-to-A or C-to-T mutations, which can be corrected via A-to-G base editing. While CRISPR-based base editors are widely used for nuclear genome editing, their application to mtDNA has been hindered by challenges in delivering guide RNAs (gRNAs) to mitochondria. Recently, transcription activator-like effector (TALE)-linked deaminases (TALEDs) have been reported, comprising TALE arrays, double-stranded DNA (dsDNA)-specific cytidine deaminase (DddA), and single-stranded DNA-specific adenosine deaminase (TadA8e). Unlike DddA-mediated C-to-T editing in mtDNA, the detailed mechanism of TALEDs for A-to-G editing remains unclear.
Recently, Professor Chen Jia’s team from the School of Life Science and Technology (SLST) at ShanghaiTech University published a study titled “Leveraging base excision repair for efficient adenine base editing of mitochondrial DNA” () in Nature Biotechnology.
This work elucidates the mechanism of TALEDs, a mitochondrial adenine
base editor, and develops enhanced tools (eTALED6s) with significantly
improved efficiency and precision, offering new avenues for treating
mitochondrial diseases.
In this study, the researchers constructed knockout cell lines for multiple base excision repair (BER)-related genes. Combined with in vitro deamination assays, they revealed that TALEDs do not directly perform A-to-G editing on dsDNA. Instead, TALEDs utilize DddA to induce C-to-U deamination, triggering the mitochondrial BER pathway. In this process, DddA in TALEDs first mediates C-to-U deamination. Cellular uracil glycosylase then excises the resulting uracil (U), generating an apurinic/apyrimidinic (AP) site. The AP site is either cleaved by APE1 or undergoes spontaneous strand breakage, forming a single-strand break (SSB). The SSB is further processed by exonuclease hMGME1 into single-stranded DNA (ssDNA), enabling TadA8e in TALEDs to mediate A-to-I deamination on ssDNA. Subsequent gap filling completes the A-to-G editing (Figure 1).
Figure 1: Schematic of the TALED working mechanism
Leveraging BER, the team developed enhanced TALEDs (eTALED6s) that significantly improved editing efficiency at target sites. By engineering the adenine deaminase TadA8e, the generated eTALED6R reduced transcriptome-wide off-target mutations, narrowed the editing window, minimized bystander effects, and enhanced editing purity. The researchers also modeled pathogenic mutations associated with Leigh syndrome and MELAS (e.g., m.A13514G) in the mitochondrial genome using eTALED6 and engineered eTALED6R (Figure 2). Oxygen consumption rate (OCR) assays confirmed that eTALEDs successfully induced the expected mitochondrial dysfunction.
Figure 2: Editing efficiency (top), product purity (middle), and transcriptome-wide off-target counts (bottom) mediated by sTALED, eTALED6, and eTALED6R at the m.13514 site in the mitochondrial genome
This study not only fills a critical gap in understanding TALED mechanisms but also establishes a suite of precise, efficient, and specific mitochondrial adenine base editors. These tools hold broad potential for modeling mitochondrial diseases, genetic correction, and foundational research.
Professor Chen Jia is the corresponding author of this paper. SLST PhD students Fan Yuhang and Xu Wenchao in Chen Jia’s group, and PhD student Gao Baoqing from Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences are the co-first authors of this paper.
