Trends and prospects in mitochondrial genome editing

Abstract

Mitochondria are of fundamental importance in programmed cell death, cellular metabolism, and intracellular calcium concentration modulation, and inheritable mitochondrial disorders via mitochondrial DNA (mtDNA) mutation cause several diseases in various organs and systems. Nevertheless, mtDNA editing, which plays an essential role in the treatment of mitochondrial disorders, still faces several challenges. Recently, programmable editing tools for mtDNA base editing, such as cytosine base editors derived from DddA (DdCBEs), transcription activator-like effector (TALE)-linked deaminase (TALED), and zinc finger deaminase (ZFD), have emerged with considerable potential for correcting pathogenic mtDNA variants. In this review, we depict recent advances in the field, including structural biology and repair mechanisms, and discuss the prospects of using base editing tools on mtDNA to broaden insight into their medical applicability for treating mitochondrial diseases.

Fig. 1. Distribution of the rate of human pathogenic mitochondrial DNA mutations.

a The overall concept of heteroplasmic shifting. Although mutant and wild-type mtDNA can coexist, the exceeded threshold of mtDNA mutations compromises oxidative phosphorylation (OXPHOS). The mutations can be corrected by using gene editing platforms, which change the heteroplasmic level toward reducing the threshold. b Pie chart demonstrating the percentage of pathogenic mitochondrial DNA mutations in the MITOMAP database (accessed November 03, 2022). The white text reveals the distribution of transition point mutations, while the black text indicates transversion point mutations, deletions, insertions, or inversions. Parentheses indicate the number of pathogenic mitochondrial DNA mutations.

Fig. 2. The architectures of several technologies for mitochondrial DNA editing.

a A schematic of the architecture of CRISPR/Cas9 in the interactions with single-guide RNA and target DNA. The structures of b mtZFN, c ZFD, d mitochondrially targeted mitoTALEN, e DdCBE, and f TALED. ZFP zinc-finger protein, MTS mitochondrial targeting sequence, NES nuclear export signal, CTD (NG) C-terminal domain with NG repeat variable diresidues, NTD N-terminal domain.

Fig. 3. Strategies for delivering gene editing platforms into mitochondria.

ZFP- and TALE-based gene editors or engineered CRISPR/Cas systems modified with MTS and stem‒loop motifs can be imported into mitochondria via transfection with AAVs or eVLPs, which encapsulate plasmid DNA or mRNA encoding the gene editing platforms. mtZFN, mitochondrial zinc-finger nuclease; TALEN transcription activator-like effector nuclease, DdCBE DddA-derived cytosine base editor, ZFD zinc-finger deaminase, TALE-linked deaminase (TALED); CRISPR/Cas clustered regularly interspaced short palindromic repeats/Cas.