Neuronal death is associated with mitochondrial dysfunction caused by mutations in mitochondrial DNA. Mitochondrial DNA becomes damaged when processes such as replication, repair, and nucleotide synthesis are compromised. This extensive accumulation of damaged mitochondrial DNA subsequently disrupts the normal function of mitochondria, leading to aging, degeneration, or even death of neurons. Mitochondrial dysfunction stands as a pivotal factor in the development of neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. Recognizing the intricate nature of their pathogenesis, there is an urgent need for more effective therapeutic interventions. In recent years, mitochondrial DNA editing tools such as zinc finger nucleases, double-stranded DNA deaminase toxin A-derived cytosine base editors, and transcription activator-like effector ligand deaminases have emerged. Their emergence will revolutionize the research and treatment of mitochondrial diseases. In this review, we summarize the advancements in mitochondrial base editing technology and anticipate its utilization in neurodegenerative diseases, offering insights that may inform preventive strategies and therapeutic interventions for disease phenotypes.

Patient-derived induced pluripotent stem cells (iPSCs) are a useful pathological model for debilitating diseases caused by mitochondrial DNA (mtDNA) mutations. We established iPSCs derived from mitochondrial disease patients, heteroplasmic for the m.3243A>G mutation. The proportion of a selected mtDNA can be reduced by delivering a programmable nuclease into the mitochondria, and we developed various mtDNA-targeted Platinum TALENs (mpTALENs) to modify m.3243A>G-iPSC heteroplasmy levels in either wild-type or mutant direction. For TALEN optimization, the use of non-conventional repeat-variable di-residues (ncRVD)-LK/WK or NM-enhanced cleavage activity and specificity, and the replacement of conventional with obligate heterodimeric FokI nuclease domains increased target specificity and protected mtDNA from copy number depletion. In vitro, depending on whether wild-type or mutant mtDNA was targeted, we could obtain m.3243A>G-iPSCs with a higher or lower mutation load, while the cells retained their ability to differentiate into three germ layers. These results demonstrate that our mpTALEN optimization created a useful tool for altering heteroplasmy levels in m.3243A>G-iPSCs, improving the potential for studying mutation pathology. The enhanced efficiency also holds promise for using m.3243G(MUT)-mpTALEN as a therapeutic strategy for treating patients suffering from m.3243A>G mitochondrial diseases.

Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled precise base substitutions and the efficient elimination of genomes carrying pathogenic mutations. However, reconstituting mtDNA deletions linked to mitochondrial myopathies remains challenging. Here, we engineered mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. Using mitochondrial EJ (mito-EJ) and mito-ScaI, we generated a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion across the full spectrum of heteroplasmy. Investigating these cells revealed a critical threshold of ∼75% deleted genomes, beyond which oxidative phosphorylation (OXPHOS) protein depletion, metabolic disruption, and impaired growth in galactose-containing media were observed. Single-cell multiomic profiling identified two distinct nuclear gene deregulation responses: one triggered at the deletion threshold and another progressively responding to heteroplasmy. Ultimately, we show that our method enables the modeling of disease-associated mtDNA deletions across cell types and could inform the development of targeted therapies.

For nearly three decades, interspecies somatic cell nuclear transfer (iSCNT) has been explored as a potential tool for cloning, regenerative medicine, and wildlife conservation. However, developmental inefficiencies remain a major challenge, largely due to persistent barriers in nucleocytoplasmic transport, mitonuclear communication, and epigenome crosstalk. This review synthesized peer-reviewed English articles from PubMed, Web of Science, and Scopus, spanning nearly three decades, using relevant keywords to explore the molecular mechanisms underlying iSCNT inefficiencies and potential improvement strategies. We highlight recent findings deepening the understanding of interspecies barriers in iSCNT, emphasizing their interconnected complexities, including the following: (1) nucleocytoplasmic incompatibility may disrupt nuclear pore complex (NPC) assembly and maturation, impairing the nuclear transport of essential transcription factors (TFs), embryonic genome activation (EGA), and nuclear reprogramming; (2) mitonuclear incompatibility could lead to nuclear and mitochondrial DNA (nDNA-mtDNA) mismatches, affecting electron transport chain (ETC) assembly, oxidative phosphorylation, and energy metabolism; (3) these interrelated incompatibilities can further influence epigenetic regulation, potentially leading to incomplete epigenetic reprogramming in iSCNT embryos. Addressing these challenges requires a multifaceted, species-specific approach that balances multiple incompatibilities rather than isolating a single factor. Gaining insight into the molecular interactions between the donor nucleus and recipient cytoplast, coupled with optimizing strategies tailored to specific pairings, could significantly enhance iSCNT efficiency, ultimately transforming experimental breakthroughs into real-world applications in reproductive biotechnology, regenerative medicine, and species conservation.

The heart requires a continuous energy supply to sustain its unceasing contraction-relaxation cycle. Mitochondria, a double-membrane organelle, generate approximately 90% of cellular energy as adenosine triphosphate (ATP) through oxidative phosphorylation, utilizing the electrochemical gradient established by the respiratory chain. Mitochondrial function is compromised by damage to mitochondrial DNA, including point mutations, deletions, duplications, or inversions. Additionally, disruptions to proteins associated with mitochondrial membranes regulating metabolic homeostasis can impair the respiratory chain's efficiency. This results in diminished ATP production and increased generation of reactive oxygen species. This review provides an overview of mutations affecting mitochondrial transporters and proteins involved in mitochondrial energy synthesis, particularly those involved in ATP synthesis and mobilization, and it examines their role in the pathogenesis of specific cardiomyopathies.

Mitochondrial DNA (mtDNA) diseases pose unique challenges for genetic counselling and require tailored approaches to address recurrence risks and reproductive options. The intricate dynamics of mtDNA segregation and heteroplasmy shift significantly impact the chances of having affected children. In addition to natural pregnancy, oocyte donation, and adoption, IVF-based approaches can reduce the risk of disease transmission. Prenatal diagnosis (PND) and preimplantation genetic testing (PGT) remain the standard methods for women carrying pathogenic mtDNA mutations; nevertheless, they are not suitable for every patient. Germline nuclear transfer (NT) has emerged as a novel therapeutic strategy, while mitochondrial gene editing has increasingly become a promising research area in the field. However, challenges and safety concerns associated with all these techniques remain, highlighting the need for long-term follow-up studies, an improved understanding of disease mechanisms, and personalized approaches to diagnosis and treatment. Given the inherent risks of adverse maternal and child outcomes, careful consideration of the balance between potential benefits and drawbacks is also warranted. This review will provide critical insights, identify knowledge gaps, and underscore the importance of advancing mitochondrial disease research in reproductive health.

Peptidomimetics, molecules that mimic the activity of natural peptides with improved stability or bioavailability, have emerged as interesting materials with applications in biomedicine. In this study, we describe a hybrid γ,γ-peptidomimetic that efficiently aims at mitochondria, a key therapeutic target associated with several disorders, in living cells. Peptide backbones with a component of cationic and hydrophobic amino acids have been shown to preferentially target mitochondria due to their high negative membrane potential and hydrophobic character of the membranous invaginations of these key organelles. We here exploit the advantageous bioorthogonal properties of a peptidomimetic scaffold that consists of an alternation of (1S,3R)-3-amino-2,2-dimethylcyclobutane-1-carboxylic acid and an Nα-functionalised cis-γ-amino-L-proline derivative. This peptidomimetic exhibited excellent membrane translocation efficiency, mitochondrial targeting ability, and biocompatibility. Mitochondrial targeting was confirmed to be dependent on the electrochemical potential generated by the electron transport chain. The presence of non-natural amino acids rendered the compound exceptionally stable in the presence of proteases, maintaining its integrity and functionality for targeting the organelle even after 1 week of incubation in serum. This stability, coupled with its targeting abilities and the low cytosolic/endosomal residual signal, facilitated the tracking of relevant mitochondrial dynamics, including fission events and intracellular movement. Additionally, this peptidomimetic scaffold allowed the sustained and precise mitochondrial targeting of a pH sensitive ratiometric probe, 5(6)-carboxy-SNARF-1, which enabled mitochondrial pH monitoring. In summary, our study introduces a biomimetic peptide with exceptional mitochondria-targeting properties, ensuring stability in biological media and offering insights into crucial mitochondrial processes.

Mitochondrial diseases represent one of the most prevalent and debilitating categories of hereditary disorders, characterized by significant genetic, biological, and clinical heterogeneity, which has driven the development of the field of engineered mitochondria. With the growing recognition of the pathogenic role of damaged mitochondria in aging, oxidative disorders, inflammatory diseases, and cancer, the application of engineered mitochondria has expanded to those non-hereditary contexts (sometimes referred to as mitochondria-related diseases). Due to their unique non-eukaryotic origins and endosymbiotic relationship, mitochondria are considered highly suitable for gene editing and intercellular transplantation, and remarkable progress has been achieved in two promising therapeutic strategies-mitochondrial gene editing and artificial mitochondrial transfer (collectively referred to as engineered mitochondria in this review) over the past two decades. Here, we provide a comprehensive review of the mechanisms and recent advancements in the development of engineered mitochondria for therapeutic applications, alongside a concise summary of potential clinical implications and supporting evidence from preclinical and clinical studies. Additionally, an emerging and potentially feasible approach involves ex vivo mitochondrial editing, followed by selection and transplantation, which holds the potential to overcome limitations such as reduced in vivo operability and the introduction of allogeneic mitochondrial heterogeneity, thereby broadening the applicability of engineered mitochondria.

The development of animal models is crucial for studying and treating mitochondrial diseases. Here we optimized adenine and cytosine deaminases to reduce off-target effects on the transcriptome and the mitochondrial genome, improving the accuracy and efficiency of our newly developed mitochondrial base editors (mitoBEs)1. Using these upgraded mitoBEs (version 2 (v2)), we targeted 70 mouse mitochondrial DNA mutations analogous to human pathogenic variants2, establishing a foundation for mitochondrial disease mouse models. Circular RNA-encoded mitoBEs v2 achieved up to 82% editing efficiency in mice without detectable off-target effects in the nuclear genome. The edited mitochondrial DNA persisted across various tissues and was maternally inherited, resulting in F1 generation mice with mutation loads as high as 100% and some mice exhibiting editing only at the target site. By optimizing the transcription activator-like effector (TALE) binding site, we developed a single-base-editing mouse model for the mt-Nd5 A12784G mutation. Phenotypic evaluations led to the creation of mouse models for the mt-Atp6 T8591C and mt-Nd5 A12784G mutations, exhibiting phenotypes corresponding to the reduced heart rate seen in Leigh syndrome and the vision loss characteristic of Leber's hereditary optic neuropathy, respectively. Moreover, the mt-Atp6 T8591C mutation proved to be more deleterious than mt-Nd5 A12784G, affecting embryonic development and rapidly diminishing through successive generations. These upgraded mitoBEs offer a highly efficient and precise strategy for constructing mitochondrial disease models, laying a foundation for further research in this field.

Mutations in mitochondrial DNA (mtDNA) can manifest phenotypically as a wide range of neuromuscular and neurodegenerative pathologies that are currently only managed symptomatically without addressing the root cause. A promising approach is the development of molecular tools aimed at mtDNA cutting or editing. Unlike nuclear DNA, a cell can have hundreds or even thousands of mitochondrial genomes, and mutations can be present either in all of them or only in a subset. Consequently, the developed tools are aimed at reducing the number of copies of mutant mtDNA or editing mutant nucleotides. Despite some progress in the field of mitochondrial genome editing in human cells, working with model animals is still limited due to the complexity of their creation. Furthermore, not all existing editing systems can be easily adapted to function within mitochondria. In this review, we evaluate the mtDNA editing tools available today, with a particular focus on specific mtDNA mutations linked to hereditary mitochondrial diseases, aiming to provide an in-depth understanding of both the opportunities and hurdles to the development of mitochondrial genome editing technologies.

Mitochondria serve as the cellular powerhouse, and their distinct DNA makes them a prospective target for gene editing to treat genetic disorders. However, the impact of genome editing on mitochondrial DNA (mtDNA) stability remains a mystery. Our study reveals previously unknown risks of genome editing that both nuclear and mitochondrial editing cause discernible transfer of mitochondrial DNA segments into the nuclear genome in various cell types including human cell lines, primary T cells, and mouse embryos. Furthermore, drug-induced mitochondrial stresses and mtDNA breaks exacerbate this transfer of mtDNA into the nuclear genome. Notably, we observe that mitochondrial editors, including mitoTALEN and recently developed base editor DdCBE, can also enhance crosstalk between mtDNA and the nuclear genome. Moreover, we provide a practical solution by co-expressing TREX1 or TREX2 exonucleases during DdCBE editing. These findings imply genome instability of mitochondria during induced DNA breaks and explain the origins of mitochondrial-nuclear DNA segments.

Leigh syndrome (LS) is a severe mitochondrial disease that results from mutations in the nuclear or mitochondrial DNA that impairs cellular respiration and ATP production. Mutations in more than 100 genes have been demonstrated to cause LS. The disease most commonly affects brain development and function, resulting in cognitive and motor impairment. The underlying pathogenesis is challenging to ascertain due to the diverse range of symptoms exhibited by affected individuals and the variability in prognosis. To understand the disease mechanisms of different LS-causing mutations and to find a suitable treatment, several different model systems have been developed over the last 30 years. This review summarizes the established disease models of LS and their key findings. Smaller organisms such as yeast have been used to study the biochemical properties of causative mutations. Drosophila melanogaster, Danio rerio, and Caenorhabditis elegans have been used to dissect the pathophysiology of the neurological and motor symptoms of LS. Mammalian models, including the widely used Ndufs4 knockout mouse model of complex I deficiency, have been used to study the developmental, cognitive, and motor functions associated with the disease. Finally, cellular models of LS range from immortalized cell lines and trans-mitochondrial cybrids to more recent model systems such as patient-derived induced pluripotent stem cells (iPSCs). In particular, iPSCs now allow studying the effects of LS mutations in specialized human cells, including neurons, cardiomyocytes, and even three-dimensional organoids. These latter models open the possibility of developing high-throughput drug screens and personalized treatments based on defined disease characteristics captured in the context of a defined cell type. By analyzing all these different model systems, this review aims to provide an overview of past and present means to elucidate the complex pathology of LS. We conclude that each approach is valid for answering specific research questions regarding LS, and that their complementary use could be instrumental in finding treatment solutions for this severe and currently untreatable disease.

Alzheimer's disease (AD) accounts for the majority of dementia cases, with aging being the primary risk factor for developing this neurodegenerative condition. Aging and AD share several characteristics, including the formation of amyloid plaques and neurofibrillary tangles, synaptic loss, and neuroinflammation. This overlap suggests that mechanisms driving the aging process might also promote AD; however, the underlying processes are not yet fully understood. In this narrative review, we will focus on the role of mitochondria, not only as the "powerhouse of the cell", but also in programmed cell death, immune response, macromolecular synthesis, and calcium regulation. We will explore both the common changes between aging and AD and the differences between them. Additionally, we will provide an overview of interventions aimed at maintaining mitochondrial function in an attempt to slow the progression of AD. This will include a discussion of antioxidant molecules, factors that trigger mitochondrial biogenesis, compounds capable of restoring the fission/fusion balance, and a particular focus on recent techniques for mitochondrial DNA gene therapy.

Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial myopathies and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of ∼75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies.

Combining prokaryotic end-joining with targeted endonucleases generates specific mtDNA deletions in human cellsEngineering a panel of cell lines with a large-scale deletion that spans the full spectrum of heteroplasmy75% heteroplasmy is the threshold that triggers mitochondrial and cellular dysfunctionTwo distinct nuclear transcriptional programs in response to mtDNA deletions: threshold-triggered and heteroplasmy-sensing.

The application of rapidly growing CRISPR toolboxes and methods has great potential to transform biomedical research. Here, we provide a snapshot of up-to-date CRISPR toolboxes, then critically discuss the promises and hurdles associated with CRISPR-based nuclear genome editing, epigenome editing, and mitochondrial editing. The technical challenges and key solutions to realize epigenome editing in vivo, in vivo base editing and prime editing, mitochondrial editing in complex tissues and animals, and CRISPR-associated transposases and integrases in targeted genomic integration of very large DNA payloads are discussed. Lastly, we discuss the latest situation of the CRISPR/Cas9 clinical trials and provide perspectives on CRISPR-based gene therapy. Apart from technical shortcomings, ethical and societal considerations for CRISPR applications in human therapeutics and research are extensively highlighted.

The role of mitochondria in neurodegenerative diseases is crucial, and recent developments have highlighted its significance in cell therapy. Mitochondrial dysfunction has been implicated in various neurodegenerative disorders, including Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, and Huntington's diseases. Understanding the impact of mitochondrial biology on these conditions can provide valuable insights for developing targeted cell therapies. This mini-review refocuses on mitochondria and emphasizes the potential of therapies leveraging mesenchymal stem cells, embryonic stem cells, induced pluripotent stem cells, stem cell-derived secretions, and extracellular vesicles. Mesenchymal stem cell-mediated mitochondria transfer is highlighted for restoring mitochondrial health in cells with dysfunctional mitochondria. Additionally, attention is paid to gene-editing techniques such as mito-CRISPR, mitoTALENs, mito-ZNFs, and DdCBEs to ensure the safety and efficacy of stem cell treatments. Challenges and future directions are also discussed, including the possible tumorigenic effects of stem cells, off-target effects, disease targeting, immune rejection, and ethical issues.

Mitochondria, mainly known as energy factories of eukaryotic cells, also exert several additional signaling and metabolic functions and are today recognized as major cellular biosynthetic and signaling hubs. Mitochondria possess their own genome (mitochondrial DNA-mtDNA), that encodes proteins essential for oxidative phosphorylation, and mutations in it are an important contributor to human disease. The mtDNA mutations often exist in heteroplasmic conditions, with both healthy and mutant versions of the mtDNA residing in patients' cells and the level of mutant mtDNA may vary between different tissues and organs and affect the clinical outcome of the disease. Thus, shifting the ratio between healthy and mutant mtDNA in patients' cells provides an intriguing therapeutic option for mtDNA diseases. In this review we describe current strategies for modulating mitochondrial heteroplasmy levels with engineered endonucleases including mitochondrially targeted TALENs and Zinc finger nucleases (ZFNs) and discuss their therapeutic potential. These gene therapy tools could in the future provide therapeutic help both for patients with mitochondrial disease as well as in preventing the transfer of pathogenic mtDNA mutations from a mother to her offspring.

Mitochondrial genome (mtDNA) independent of nuclear gene is a set of double-stranded circular DNA that encodes 13 proteins, 2 ribosomal RNAs and 22 mitochondrial transfer RNAs, all of which play vital roles in functions as well as behaviors of mitochondria. Mutations in mtDNA result in various mitochondrial disorders without available cures. However, the manipulation of mtDNA via the mitochondria-targeted gene delivery faces formidable barriers, particularly owing to the mitochondrial double membrane. Given the fact that there are various transport channels on the mitochondrial membrane used to transfer a variety of endogenous substances to maintain the normal functions of mitochondria, mitochondrial endogenous substance transport-inspired nanomaterials have been proposed for mitochondria-targeted gene delivery. In this review, we summarize mitochondria-targeted gene delivery systems based on different mitochondrial endogenous substance transport pathways. These are categorized into mitochondrial steroid hormones import pathways-inspired nanomaterials, protein import pathways-inspired nanomaterials and other mitochondria-targeted gene delivery nanomaterials. We also review the applications and challenges involved in current mitochondrial gene editing systems. This review delves into the approaches of mitochondria-targeted gene delivery, providing details on the design of mitochondria-targeted delivery systems and the limitations regarding the various technologies. Despite the progress in this field is currently slow, the ongoing exploration of mitochondrial endogenous substance transport and mitochondrial biological phenomena may act as a crucial breakthrough in the targeted delivery of gene into mitochondria and even the manipulation of mtDNA.

The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA-targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR-associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD.

One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts. Here we aimed to further explore how target composition and TALEB architecture will impact the editing outcomes. We demonstrated how the nature of the linker between TALE array and split DddAtox head allows us to fine tune the editing window, also controlling possible bystander activity. Furthermore, we showed that both the TALEB architecture and spacer length separating the two TALE DNA binding regions impact the target TC editing dependence by the surrounding bases, leading to more restrictive or permissive editing profiles.

Mitochondria are subcellular organelles essential for life. Beyond their role in producing energy, mitochondria govern various physiological mechanisms, encompassing energy generation, metabolic processes, apoptotic events, and immune responses. Mitochondria also contain genetic material that is susceptible to various forms of damage. Mitochondrial double-stranded breaks (DSB) are toxic lesions that the nucleus repairs promptly. Nevertheless, the significance of DSB repair in mammalian mitochondria is controversial. This review presents an updated view of the available research on the consequences of mitochondrial DNA DSB from the molecular to the cellular level. We discuss the crucial function of mitochondrial DNA damage in regulating processes such as senescence, integrated stress response, and innate immunity. Lastly, we discuss the potential role of mitochondrial DNA DSB in mediating the cellular consequences of ionizing radiations, the standard of care in treating solid tumors.

The manipulation of animal mitochondrial genomes has long been a challenge due to the lack of an effective transformation method. With the discovery of specific gene editing enzymes, designed to target pathogenic mitochondrial DNA mutations (often heteroplasmic), the selective removal or modification of mutant variants has become a reality. Because mitochondria cannot efficiently import RNAs, CRISPR has not been the first choice for editing mitochondrial genes. However, the last few years witnessed an explosion in novel and optimized non-CRISPR approaches to promote double-strand breaks or base-edit of mtDNA in vivo. Engineered forms of specific nucleases and cytidine/adenine deaminases form the basis for these techniques. I will review the newest developments that constitute the current toolbox for animal mtDNA gene editing in vivo, bringing these approaches not only to the exploration of mitochondrial function, but also closer to clinical use.

Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria, yet they maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures and undergoing frequent homologous recombination. To date, it has proven to be challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene-editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially transcription activator-like effector nucleases (TALENs) and artificial enzymes utilizing DNA-binding domains of TALENs (TALEs), have been successfully used to modify these genomes by harnessing organellar-targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors in organellar genomes, their effects and their potential applications in plant science and breeding.

Introduction: Mitochondrial diseases caused by mtDNA have no effective cures. Recently developed DddA-derived cytosine base editors (DdCBEs) have potential therapeutic implications in rescuing the mtDNA mutations. However, the performance of DdCBEs relies on designing different targets or improving combinations of split-DddA halves and orientations, lacking knowledge of predicting the results before its application. Methods: A series of DdCBE pairs for wide ranges of aC or tC targets was constructed, and transfected into Neuro-2a cells. The mutation rate of targets was compared to figure out the potential editing rules. Results: It is found that DdCBEs mediated mtDNA editing is predictable: 1) aC targets have a concentrated editing window for mtDNA editing in comparison with tC targets, which at 5'C8-11 (G1333) and 5'C10-13 (G1397) for aC target, while 5'C4-13 (G1333) and 5'C5-14 (G1397) for tC target with 16bp spacer. 2) G1333 mediated C>T conversion at aC targets in DddA-half-specific manner, while G1333 and G1397 mediated C>T conversion are DddA-half-prefer separately for tC and aC targets. 3) The nucleotide adjacent to the 3' end of aC motif affects mtDNA editing. Finally, by the guidance of these rules, a cell model harboring a pathogenic mtDNA mutation was constructed with high efficiency and no bystander effects. Discussion: In summary, this discovery helps us conceive the optimal strategy for accurate mtDNA editing, avoiding time- and effort-consuming optimized screening jobs.

Plastids and mitochondria are 2 intracellular organelles containing DNA-encoding partial but essential components for their roles, photosynthesis, and respiration. Precise base editing in both plastid and mitochondrial genomes would benefit their gene functional analysis and crop breeding. Targeted base editing in organellar genomes relies on a protein-based genome-editing system that uses the TALE-DNA recognition motif with deaminases. This is because the efficient delivery of guide RNA for clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems into organelles is currently impossible. Since TALE-based base editors used in organellar genomes are usually dimeric types, in this study, we used targeted A-to-G base editing in Arabidopsis (Arabidopsis thaliana) plastid and mitochondrial genomes with monomeric TALE-based deaminase for easier assembling of vectors. As a result, inheritable targeted A-to-G base editing of adenosine triphosphatase subunit 6-2 (atp6-2) in plant mitochondrial genomes and of 16S ribosomal RNA (16S rRNA) in plastid genomes of Arabidopsis was successfully induced by monomeric TALE-based adenine deaminase (AD) without off-target mutations. The monomeric TALE-based adenine deaminases also demonstrated a preference for editing the 8th T on the same strand from the recognition end. Phenotypic analysis showed that A-to-G conversion at 1139A of plastid 16S rRNA conferred substantial spectinomycin resistance in Arabidopsis, but not the other 2 potential-resistant mutations at 1131T and 1137T, predicted from the previous bacterial data. Our study demonstrated the feasibility of monomeric TALE-based ADs in plant organelles and their potential contribution to the functional analyses of plant organelles with easier assembling.

Mutations within mtDNA frequently give rise to severe encephalopathies. Given that a majority of these mtDNA defects exist in a heteroplasmic state, we harnessed the precision of mitochondrial-targeted TALEN (mitoTALEN) to selectively eliminate mutant mtDNA within the CNS of a murine model harboring a heteroplasmic mutation in the mitochondrial tRNA alanine gene (m.5024C>T). This targeted approach was accomplished by the use of AAV-PHP.eB and a neuron-specific synapsin promoter for effective neuronal delivery and expression of mitoTALEN. We found that most CNS regions were effectively transduced and showed a significant reduction in mutant mtDNA. This reduction was accompanied by an increase in mitochondrial tRNA alanine levels, which are drastically reduced by the m.5024C>T mutation. These results showed that mitochondrial-targeted gene editing can be effective in reducing CNS-mutant mtDNA in vivo, paving the way for clinical trials in patients with mitochondrial encephalopathies.

TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.

Mitochondrial gene editing holds great promise as a therapeutic approach for mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA). Current strategies focus on reducing mutant mtDNA heteroplasmy levels through targeted cleavage or base editing. However, the delivery of editing components into mitochondria remains a challenge. Here we investigate the import of CRISPR-Cas12a system guide RNAs (crRNAs) into human mitochondria and study the structural requirements for this process by northern blot analysis of RNA isolated from nucleases-treated mitoplasts. To investigate whether the fusion of crRNA with known RNA import determinants (MLS) improve its mitochondrial targeting, we added MLS hairpin structures at 3'-end of crRNA and demonstrated that this did not impact crRNA ability to program specific cleavage of DNA in lysate of human cells expressing AsCas12a nuclease. Surprisingly, mitochondrial localization of the fused crRNA molecules was not improved compared to non-modified version, indicating that structured scaffold domain of crRNA can probably function as MLS, assuring crRNA mitochondrial import. Then, we designed a series of crRNAs targeting different regions of mtDNA and demonstrated their ability to program specific cleavage of mtDNA fragments in cell lysate and their partial localization in mitochondrial matrix in human cells transfected with these RNA molecules. We hypothesize that mitochondrial import of crRNAs may depend on their secondary structure/sequence. We presume that imported crRNA allow reconstituting the active crRNA/Cas12a system in human mitochondria, which can contribute to the development of effective strategies for mitochondrial gene editing and potential future treatment of mitochondrial diseases.

Mitochondrial DNA (mtDNA), a multicopy genome found in mitochondria, is crucial for oxidative phosphorylation. Mutations in mtDNA can lead to severe mitochondrial dysfunction in tissues and organs with high energy demand. MtDNA mutations are closely associated with mitochondrial and age-related disease. To better understand the functional role of mtDNA and work toward developing therapeutics, it is essential to advance technology that is capable of manipulating the mitochondrial genome. This review discusses ongoing efforts in mitochondrial genome editing with mtDNA nucleases and base editors, including the tools, delivery strategies, and applications. Future advances in mitochondrial genome editing to address challenges regarding their efficiency and specificity can achieve the promise of therapeutic genome editing. [BMB Reports 2024; 57(1): 19-29].

Mitochondrial DNA (mtDNA) is a critical genome contained within the mitochondria of eukaryotic cells, with many copies present in each mitochondrion. Mutations in mtDNA often are inherited and can lead to severe health problems, including various inherited diseases and premature aging. The lack of efficient repair mechanisms and the susceptibility of mtDNA to damage exacerbate the threat to human health. Heteroplasmy, the presence of different mtDNA genotypes within a single cell, increases the complexity of these diseases and requires an effective editing method for correction. Recently, gene-editing techniques, including programmable nucleases such as restriction endonuclease, zinc finger nuclease, transcription activator-like effector nuclease, clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated 9 and base editors, have provided new tools for editing mtDNA in mammalian cells. Base editors are particularly promising because of their high efficiency and precision in correcting mtDNA mutations. In this review, we discuss the application of these techniques in mitochondrial gene editing and their limitations. We also explore the potential of base editors for mtDNA modification and discuss the opportunities and challenges associated with their application in mitochondrial gene editing. In conclusion, this review highlights the advancements, limitations and opportunities in current mitochondrial gene-editing technologies and approaches. Our insights aim to stimulate the development of new editing strategies that can ultimately alleviate the adverse effects of mitochondrial hereditary diseases.

In this review, we detail the current state of application of gene therapy to primary mitochondrial disorders (PMDs). Recombinant adeno-associated virus-based (rAAV) gene replacement approaches for nuclear gene disorders have been undertaken successfully in more than ten preclinical mouse models of PMDs which has been made possible by the development of novel rAAV technologies that achieve more efficient organ targeting. So far, however, the greatest progress has been made for Leber Hereditary Optic Neuropathy, for which phase 3 clinical trials of lenadogene nolparvovec demonstrated efficacy and good tolerability. Other methods of treating mitochondrial DNA (mtDNA) disorders have also had traction, including refinements to nucleases that degrade mtDNA molecules with pathogenic variants, including transcription activator-like effector nucleases, zinc-finger nucleases, and meganucleases (mitoARCUS). rAAV-based approaches have been used successfully to deliver these nucleases in vivo in mice. Exciting developments in CRISPR-Cas9 gene editing technology have achieved in vivo gene editing in mouse models of PMDs due to nuclear gene defects and new CRISPR-free gene editing approaches have shown great potential for therapeutic application in mtDNA disorders. We conclude the review by discussing the challenges of translating gene therapy in patients both from the point of view of achieving adequate organ transduction as well as clinical trial design.

Mitochondrial base editing with DddA-derived cytosine base editor (DdCBE) is limited in the accessible target sequences and modest activity. Here, the optimized DdCBE tools is presented with improved editing activity and expanded C-to-T targeting scope by fusing DddA11 variant with different cytosine deaminases with single-strand DNA activity. Compared to previous DdCBE based on DddA11 variant alone, fusion of the activation-induced cytidine deaminase (AID) from Xenopus laevis not only permits cytosine editing of 5'-GC-3' sequence, but also elevates editing efficiency at 5'-TC-3', 5'-CC-3', and 5'-GC-3' targets by up to 25-, 10-, and 6-fold, respectively. Furthermore, the A-to-G editing efficiency is significantly improved by fusing the evolved DddA6 variant with TALE-linked deoxyadenosine deaminase (TALED). Notably, the authors introduce the reported high-fidelity mutations in DddA and add nuclear export signal (NES) sequences in DdCBE and TALED to reduce off-target editing in the nuclear and mitochondrial genome while improving on-target editing efficiency in mitochondrial DNA (mtDNA). Finally, these engineered mitochondrial base editors are shown to be efficient in installing mtDNA mutations in human cells or mouse embryos for disease modeling. Collectively, the study shows broad implications for the basic study and therapeutic applications of optimized DdCBE and TALED.

Mitochondria and chloroplasts are organelles that include their own genomes, which encode key genes for ATP production and carbon dioxide fixation, respectively. Mutations in mitochondrial DNA can cause diverse genetic disorders and are also linked to ageing and age-related diseases, including cancer. Targeted editing of organellar DNA should be useful for studying organellar genes and developing novel therapeutics, but it has been hindered by lack of efficient tools in living cells. Recently, CRISPR-free, protein-only base editors, such as double-stranded DNA deaminase toxin A-derived cytosine base editors (DdCBEs) and adenine base editors (ABEs), have been developed, which enable targeted organellar DNA editing in human cell lines, animals and plants. In this Review, we present programmable deaminases developed for base editing of organellar DNA in vitro and discuss mitochondrial DNA editing in animals, and plastid genome (plastome) editing in plants. We also discuss precision and efficiency limitations of these tools and propose improvements for therapeutic, agricultural and environmental applications.

Leber's hereditary optic neuropathy (LHON) is a common mitochondrial genetic disease, causing irreversible blindness in young individuals. Current treatments are inadequate, and there is no definitive cure. This study evaluates the effectiveness of delivering wildtype human NADH ubiquinone oxidoreductase subunit 4 (hND4) gene using mito-targeted AAV(MTSAAV) to rescue LHOH mice. We observed a declining pattern in electroretinograms amplitudes as mice aged across all groups (p < 0.001), with significant differences among groups (p = 0.023; Control vs. LHON, p = 0.008; Control vs. Rescue, p = 0.228). Inner retinal thickness and intraocular pressure did not change significantly with age or groups. Compared to LHON mice, those rescued with wildtype hND4 exhibited improved retinal visual acuity (0.29 ± 0.1 cy/deg vs. 0.15 ± 0.1 cy/deg) and increased functional hyperemia response (effect of flicker, p < 0.001, effect of Group, p = 0.004; Interaction Flicker × Group, p < 0.001). Postmortem analysis shows a marked reduction in retinal ganglion cell density in the LHON group compared to the other groups (Effect of Group, p < 0.001, Control vs. LHON, p < 0.001, Control vs. Rescue, p = 0.106). These results suggest that MTSAAV-delivered wildtype hND4 gene rescues, at least in part, visual impairment in an LHON mouse model and has the therapeutic potential to treat this disease.

Inflammatory bowel disease (IBD) is a prototypic complex disease in the gastrointestinal tract that has been increasing in incidence and prevalence in recent decades. Although the precise pathophysiology of IBD remains to be elucidated, a large body of evidence suggests the critical roles of mitochondria and intestinal microbiota in the pathogenesis of IBD. In addition to their contributions to the disease, both mitochondria and gut microbes may interact with each other and modulate disease-causing cell activities. Therefore, we hypothesize that dissecting this unique interaction may help to identify novel pathways involved in IBD, which will further contribute to discovering new therapeutic approaches to the disease. As poorly treated IBD significantly affects the quality of life of patients and is associated with risks and complications, successful treatment is crucial. In this review, we stratify previously reported experimental and clinical observations of the role of mitochondria and intestinal microbiota in IBD. Additionally, we review the intercommunication between mitochondria, and the intestinal microbiome in patients with IBD is reviewed along with the potential mediators for these interactions. We specifically focus on their roles in cellular metabolism in intestinal epithelial cells and immune cells. To this end, we propose a potential therapeutic intervention strategy for IBD.

The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. cerevisiae mitochondria can exist with partial (ρ- mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well-suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome.

Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.

Pathogenic mutations in mitochondrial DNA cause severe and often lethal multi-system symptoms in primary mitochondrial defects. However, effective therapies for these defects are still lacking. Strategies such as employing mitochondrially targeted restriction enzymes or programmable nucleases to shift the ratio of heteroplasmic mutations and allotopic expression of mitochondrial protein-coding genes have limitations in treating mitochondrial homoplasmic mutations, especially in non-coding genes. Here, we conduct a proof of concept study applying a screened DdCBE pair to correct the homoplasmic m.A4300G mutation in induced pluripotent stem cells derived from a patient with hypertrophic cardiomyopathy. We achieve efficient G4300A correction with limited off-target editing, and successfully restore mitochondrial function in corrected induced pluripotent stem cell clones. Our study demonstrates the feasibility of using DdCBE to treat primary mitochondrial defects caused by homoplasmic pathogenic mitochondrial DNA mutations.

Allotopic expression is the functional transfer of an organellar gene to the nucleus, followed by synthesis of the gene product in the cytosol and import into the appropriate organellar sub compartment. Here, we focus on mitochondrial genes encoding OXPHOS subunits that were naturally transferred to the nucleus, and critically review experimental evidence that claim their allotopic expression. We emphasize aspects that may have been overlooked before, i.e., when modifying a mitochondrial gene for allotopic expression━besides adapting the codon usage and including sequences encoding mitochondrial targeting signals━three additional constraints should be considered: (i) the average apparent free energy of membrane insertion (μΔGapp) of the transmembrane stretches (TMS) in proteins earmarked for the inner mitochondrial membrane, (ii) the final, functional topology attained by each membrane-bound OXPHOS subunit; and (iii) the defined mechanism by which the protein translocator TIM23 sorts cytosol-synthesized precursors. The mechanistic constraints imposed by TIM23 dictate the operation of two pathways through which alpha-helices in TMS are sorted, that eventually determine the final topology of membrane proteins. We used the biological hydrophobicity scale to assign an average apparent free energy of membrane insertion (μΔGapp) and a "traffic light" color code to all TMS of OXPHOS membrane proteins, thereby predicting which are more likely to be internalized into mitochondria if allotopically produced. We propose that the design of proteins for allotopic expression must make allowance for μΔGapp maximization of highly hydrophobic TMS in polypeptides whose corresponding genes have not been transferred to the nucleus in some organisms.

Mitochondrial DNA (mtDNA) mutations result in a variety of genetic diseases. As an emerging therapeutic method, mtDNA editing technology recognizes targets more based on the protein and less on the nucleic acid. Although the protein recognition type mtDNA editing technology represented by zinc finger nuclease technology, transcription activator like effector nuclease technology and base editing technology has made some progress, the disadvantages of complex recognition sequence design hinder further popularization. Gene editing based on nucleic acid recognition by the CRISPR system shows superiority due to the simple structure, easy design and modification. However, the lack of effective means to deliver nucleic acids into mitochondria limits application in the field of mtDNA editing. With the advances in the study of endogenous and exogenous import pathways and the deepening understanding of DNA repair mechanisms, growing evidence shows the feasibility of nucleic acid delivery and the broad application prospects of nucleic acid recognition type mtDNA editing technology. Based on the classification of recognition elements, this article summarizes the current principles and development of mitochondrial gene editing technology, and discusses its application prospects.

Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.

Mitochondrial DNA (mtDNA) replication stalling is considered an initial step in the formation of mtDNA deletions that associate with genetic inherited disorders and aging. However, the molecular details of how stalled replication forks lead to mtDNA deletions accumulation are still unclear. Mitochondrial DNA deletion breakpoints preferentially occur at sequence motifs predicted to form G-quadruplexes (G4s), four-stranded nucleic acid structures that can fold in guanine-rich regions. Whether mtDNA G4s form in vivo and their potential implication for mtDNA instability is still under debate. In here, we developed new tools to map G4s in the mtDNA of living cells. We engineered a G4-binding protein targeted to the mitochondrial matrix of a human cell line and established the mtG4-ChIP method, enabling the determination of mtDNA G4s under different cellular conditions. Our results are indicative of transient mtDNA G4 formation in human cells. We demonstrate that mtDNA-specific replication stalling increases formation of G4s, particularly in the major arc. Moreover, elevated levels of G4 block the progression of the mtDNA replication fork and cause mtDNA loss. We conclude that stalling of the mtDNA replisome enhances mtDNA G4 occurrence, and that G4s not resolved in a timely manner can have a negative impact on mtDNA integrity.