The fundamental mechanism shared by these genome-editing platforms revolves around the induction of DNA double-strand breaks (DSBs) at specific genomic loci. ZFNs and TALENs employ customizable DNA-binding domains fused to nuclease components, enabling the targeted cleavage of disease-associated genes. CRISPR–Cas systems, leveraging RNA-guided endonucleases, have dramatically simplified the editing process, providing ease of design and multiplexing capabilities. Yet, despite their promise, reliance on DSBs introduces challenges related to error-prone repair pathways such as non-homologous end joining (NHEJ), which may cause unwanted insertions or deletions and off-target mutagenesis, potentially jeopardizing safety profiles in therapeutic contexts.

Addressing these concerns, recent advancements have extended genome editing beyond DSB-dependent repair mechanisms. Base editors represent a landmark innovation that allows direct conversion of one nucleotide to another without cleaving the DNA backbone. By fusing catalytically impaired Cas proteins with deaminase enzymes, these editors offer refined correction of point mutations, which constitute the majority of pathogenic variants. Complementarily, prime editors utilize a reverse transcriptase fused to a Cas nickase guided by a prime editing RNA to precisely insert, delete, or replace small DNA sequences. These technologies expand the range of editable mutations, minimize genotoxic stress, and facilitate applications in cells less amenable to conventional genome-editing processes.

Concurrent with innovations in editing enzymes, the delivery of editing cargo into target cells has evolved with equal rapidity. Various formats—including plasmid DNA, viral-vector DNA, mRNA, guide RNA molecules, and ribonucleoprotein (RNP) complexes—have been optimized to balance efficiency, transient expression, and safety. Viral vectors such as adeno-associated viruses (AAVs), adenoviruses, and herpesviruses offer high transduction efficiency and tropism for specific tissues but face challenges related to immunogenicity, packaging size constraints, and long-term expression control. Non-viral modalities, such as lipid nanoparticles (LNPs), electroporation, and virus-like particles, provide alternative strategies to deliver editing components with reduced immune activation and enhanced scalability for clinical manufacturing.

The utility of genome-editing technologies extends beyond simple gene knockout or correction. Endogenous gene tagging using genome editing allows for in situ labeling of proteins, enabling dynamic functional studies under physiological conditions. High-throughput functional variant screening leverages CRISPR libraries to decipher the phenotypic consequences of millions of genetic alterations, accelerating the discovery of clinically relevant genomic elements. Additionally, genome editing has opened novel avenues such as molecular recording within cells, where programmable edits serve as chronological logs responding to biological stimuli, facilitating the understanding of complex biological processes with unprecedented resolution. These applications underscore the transformative potential of genome editing in both research and therapeutic contexts.

Clinically, these technologies have rapidly advanced into therapeutic pipelines for a broad array of diseases. Oncology has benefited from engineered cellular immunotherapies, including chimeric antigen receptor (CAR) T cells modified via genome editing to enhance tumor targeting, persistence, and safety. Hematology is witnessing breakthroughs in editing hematopoietic stem and progenitor cells to correct hemoglobinopathies such as sickle cell disease and beta-thalassemia. Liver-directed genome editing programs, targeting metabolic and coagulation disorders, capitalize on the organ’s regenerative capacity and accessibility, marking a critical step in treating diseases that were previously intractable.

Despite these successes, significant challenges remain before genome editing can achieve widespread clinical viability. Enhancing editing fidelity to minimize off-target effects remains a top priority, as unintended genomic alterations can lead to oncogenesis or other adverse outcomes. Broadening the protospacer adjacent motif (PAM) flexibility of Cas enzymes is equally crucial, widening the spectrum of accessible genomic sites and thereby expanding therapeutic applicability. Furthermore, efficient editing of non-dividing cells, such as neurons, is essential for addressing neurodegenerative diseases but remains technically demanding due to limitations in DNA repair pathways active in these cells.

Another domain of active investigation is in tissue-selective delivery. Certain organs, notably the lung and central nervous system, present formidable barriers to therapeutic delivery due to their anatomical complexity and protective physiological barriers like the blood-brain barrier. Innovations in vector engineering, nanoparticles specifically designed to traverse these barriers, and localized administration routes are essential to realizing effective in vivo genome editing in these challenging tissues. Moreover, refining delivery methods to achieve cell-type specificity will mitigate off-tissue effects, a paramount consideration for clinical safety.

Manufacturing scalability and standardization are equally imperative for the clinical translation of genome-editing therapeutics. The production of clinical-grade editing components under good manufacturing practice (GMP) conditions requires reproducible protocols, quality control, and cost-effective platforms to ensure widespread access. Additionally, long-term patient monitoring frameworks must be established to identify delayed adverse effects, potential immune responses, and durable therapeutic outcomes. Importantly, addressing socioeconomic disparities and enabling equitable access globally constitute ethical imperatives as genome editing enters mainstream healthcare.

As genome editing technologies mature, integration with complementary therapeutic strategies will likely enhance their clinical impact. For example, combining genome editing with gene therapy vectors, epigenetic modulators, or immune checkpoint inhibitors could synergistically improve treatment efficacy. Additionally, synthetic biology frameworks may incorporate editing capabilities within cellular circuits, enabling programmable cellular behaviors responsive to disease states. The convergence of these fields promises an era of truly precision medicine tailored to individual genetic landscapes and disease mechanisms.

From a regulatory perspective, genome editing poses unique challenges that regulatory agencies worldwide are actively tackling. Assessing the safety and efficacy of these modalities necessitates novel frameworks that account for the permanent alterations introduced at the genomic level. Post-marketing surveillance measures and robust patient registries will play integral roles in safeguarding public health while accelerating access to life-saving interventions. Collaboration between scientists, clinicians, regulators, and patient advocacy groups will be essential to harmonize guidelines and expedite responsible translation.

In summary, genome editing technologies represent a paradigm shift with enormous potential for the treatment of a vast spectrum of human diseases. The continuous refinement of programmable nucleases, base and prime editors, coupled with advances in delivery technologies and clinical strategies, are propelling this field from experimental therapy to routine clinical application. While obstacles relating to precision, delivery, safety, and accessibility remain, rapid scientific progress and interdisciplinary collaboration offer a compelling vision of a future where many currently incurable conditions may be effectively treated or even cured through the power of genome editing.

Subject of Research: Genome editing technologies and their application in the treatment of human diseases.

Article Title: Applications of genome editing technologies in the treatment of human diseases.

Article References:
Alshorman, J., Mehran, M.J., Miyanda Tembo, K. et al. Applications of genome editing technologies in the treatment of human diseases. Gene Ther (2026). https://doi.org/10.1038/s41434-026-00627-z

Image Credits: AI Generated

DOI: 15 June 2026

In recent years, a range of mitochondrial base editors (BEs) emerged, including DddA-derived cytosine base editors (DdCBEs) and TALE-linked deaminases (TALEDs). Our own laboratory introduced a new type of mitochondrial base editor, abbreviated mitoBE, capable of cytosine-to-thymine (C-to-T) and adenine-to-guanine (A-to-G) conversions in the mitochondrial genome. These constructs combine a double-stranded or single-stranded DNA deaminase with a DNA nickase, harnessing the potential of transcription activator-like effectors (TALEs) that are customized to bind specific sequences in mtDNA. The original version of mitoBEs, described previously, showed minimal off-target editing, primarily because the TALE design confers strong strand specificity. Nonetheless, the fervent push for generating mouse models to replicate the precise mutations implicated in human mitochondrial disorders called for an even higher level of accuracy and efficiency, particularly when microinjecting these tools as RNA into zygotes.

The next-generation variant, termed mitoBEs v2, arose from the thorough redesign of adenine and cytosine deaminases to mitigate unwanted off-target edits. When expressed in cells, the original versions of these mitochondrial base editors were shown to cause occasional undesired edits in the mitochondrial genome and, more significantly, in transcripts, because certain cytosine and adenine deaminases can inadvertently bind and deaminate RNA. This risk becomes more pronounced when the editors are delivered in the form of mRNA, which yields higher expression levels and correspondingly higher on-target editing but also riskier interactions with off-target substrates. By strategically mutating and screening key residues within TadA, a bacterial adenine deaminase commonly used for adenine base editing, the new version of mitoABE included the substitution V28F in TadA8e-V106W, leading to substantially improved specificity and reduced RNA off-target edits. In parallel, our team explored a library of cytosine deaminases for an alternative to APOBEC1—the widely used but often promiscuous deaminase—ultimately selecting CBE6d, a TadA-derived cytosine deaminase that delivered a higher degree of efficiency and a narrower editing window.

Having validated mitoBEs v2 in cultured cells, we systematically screened 70 pathogenic point mutations in the mouse mitochondrial genome that mirror known human mutations. Among these, we found 68 to be editable by mitoBEs v2, indicating that the modified editors maintain broad applicability for disease modeling. The editing efficiency often exceeded 10%, and in some loci, including mt-Rnr1 A978G, mt-TrnV G1029A, mt-Atp6 T8576C, mt-Atp6 T8591C, mt-Nd5 T12499C, and mt-Nd5 A12784G, we observed rates of up to 20–25% in cultured neuroblastoma cells. While such cell-line testing provides only an initial screening for the feasibility of each target site, it also reveals the interplay between TALE binding, deaminase activity, and the architecture of the target sequence in shaping how effectively any given site can be mutated.

The next challenge was to translate this high efficiency to live animals, where editing has to happen at the zygote stage so that every cell in the adult mouse carries the mtDNA change, making it possible to recapitulate human disease phenotypes. We chose two specific positions for in vivo proof-of-concept studies: mt-Atp6 T8591C, corresponding to the human m.T9191C mutation implicated in Leigh syndrome, and mt-Nd5 A12784G, mirroring the human m.A13379G mutation implicated in LHON. When we delivered mRNA or circRNA encoding the new mitoBEs v2 into one-cell mouse embryos, the editing efficiencies reached unprecedented levels. Notably, circRNA-encoded mitoBEs v2 proved more effective than mRNA-encoded versions, often doubling the mutation rates. In some embryos, we attained editing efficiency upward of 60% at T8591C or 62% at A12784G when analyzed at the blastocyst stage. Extending this success to live offspring, many F0 mice carried mutation loads of 40–50% or even up to 82% at their respective loci, underscoring the ability of mitoBEs v2 to create highly heteroplasmic or near-homoplasmic conditions.

This level of editing in F0 mice is particularly important for studying mitochondrial disorders because disease phenotypes often manifest only when the proportion of mutated mtDNA surpasses a threshold. In LHON, for instance, the typical threshold is around 60% for the mutant mtDNA to induce the visual impairments commonly observed in patients, though lower loads can sometimes be sufficient. By generating mice in which the majority of their mtDNA is mutated, we produce more faithful analogs of human disease states, facilitating a clearer understanding of pathological mechanisms and the development of treatments. Importantly, we verified the specificity of these edits. Whole-genome sequencing revealed no significant off-target editing within the nuclear genome at relevant sequencing depths, and a thorough survey of potential off-target sites, including computational predictions with TALENoffer, showed the background to be effectively clean. Even in the mitochondrial genome, where the original version of mitoCBEs occasionally introduced off-target conversions, the new cytosine deaminase variant CBE6d displayed minimal detectable bystander or off-target edits.

One of the most intriguing discoveries about these newly engineered mice was the extent to which the mutations were stably propagated across various somatic tissues and over time. By sampling 26 tissues at 2 months of age in two different F0 mice, we saw that editing levels remained relatively stable in many tissues. Some variations were detected, which could be due to the interplay between environmental factors, tissue-specific energetic demands, and potential selective pressures on certain mtDNA variants. Indeed, such tissue-specific segregation is part of the broader story of the mitochondrial genetic bottleneck, wherein different subpopulations of mtDNA can get amplified or suppressed depending on energetic or developmental constraints.

The question of heritability was addressed by mating female mice carrying the edited mtDNA with wild-type males. mtDNA is well known to be maternally inherited in mammals. We observed that mutation loads fluctuated in F1 and F2 generations, sometimes increasing, sometimes decreasing, a classic manifestation of the mitochondrial bottleneck effect. Remarkably, some F1 offspring attained 100% mutation load at the A12784G site, showcasing the potential to generate fully homoplasmic lines within just one generation. In contrast, the T8591C variants seemed detrimental to embryonic development or gamete maturation, as illustrated by lower birth rates in those lines and a gradual diminution of the T8591C mutation over subsequent generations. By the F2 or F3 generation, T8591C either was substantially reduced or disappeared in most offspring, pointing to a powerful selective force that eliminates highly deleterious mtDNA variants.

These lineage-tracking observations in mice are emblematic of the broader phenomenon experienced by human carriers of pathogenic mtDNA. Some families are known to spontaneously lose or reduce detrimental variants over generations, presumably because these variants compromise oocyte or embryo viability. Conversely, other variants, even if pathogenic, can persist or expand under certain circumstances. The ability to control and quantify these effects in a laboratory mouse population is an invaluable asset for dissecting exactly how mitochondrial heteroplasmy shifts occur, what molecular signals lead to selection for or against certain mtDNA haplotypes, and how to potentially intervene in disease scenarios.

Phenotypic characterization of the F0 mice revealed disease-relevant symptoms that mirror clinical data in humans. Mice with high editing levels at T8591C in mt-Atp6 had significantly reduced heart rates and a notably diminished left ventricular ejection fraction as assessed by echocardiography. Leigh syndrome is often associated with severe cardiovascular and neurological abnormalities. The phenotypic data in these mice strengthen the link between that particular point mutation and the observed phenotype, consistent with human clinical observations that T9191C can lead to Leigh syndrome with pronounced cardiac manifestations. Similarly, the mice carrying the A12784G mutation in mt-Nd5 displayed impaired visual function evidenced by electroretinography. Under dark-adapted conditions, both a-wave and b-wave responses were suppressed in these animals, and under light-adaptation conditions, the b-wave responses were notably depressed. LHON in humans is characterized by central vision loss, optic atrophy, and deficits in the photoreceptor signals, which aligns with these findings. By recapitulating such phenotypes, these new mouse lines represent crucial platforms for future interventions, drug testing, and mechanistic analyses of mitochondrial diseases.

Despite these accomplishments, a challenge remains in achieving truly single-base alterations without introducing secondary edits in the adjacent window. Because TALE-based editors typically incorporate a small window in which deamination can occur, multiple bases within that window can be converted if the sequence context permits. While the presence of bystander mutations is tolerable in certain therapeutic contexts, disease modeling demands the highest precision to unambiguously link genotype to phenotype. In the present study, we addressed this by shifting the TALE-binding sites in the mt-Nd5 A12784G system, effectively narrowing the potential editing window so that only the base at position 12784 is selectively edited. Indeed, some screening in cell lines identified pairs of TALE monomers that yield cleaner edits, and embryo injections of these improved pairs gave rise to F0 mice whose only edit was at the target site. Phenotypic tests using electroretinography on these single-mutation mice confirmed that even in the absence of neighboring bystander mutations, the A12784G change was sufficient to cause LHON-like visual defects. Therefore, rational design of TALE binding, coupled with the improved catalytic specificity of deaminases, can yield nearly perfect single-base mitochondrial edits.

The success of mitoBEs v2 in generating robust mouse models of disease points the way to further possibilities in therapeutic development. Gene therapy approaches using adeno-associated virus or lipid-based nanoparticle systems could, in principle, deliver these editors to adult tissues. However, the inefficiency of delivering proteins or RNAs specifically to mitochondria in vivo remains a formidable obstacle. The impetus to solve such challenges is growing, given that more than 90 disease-related point mutations in human mtDNA have been identified. Of these, around 85 are theoretically addressable by some form of base editor that converts A-to-G or C-to-T. Although the present study focuses on using mitoBEs v2 to produce heritable changes in mouse zygotes, one can envision analogous methods, refined delivery vehicles, or direct in vivo injections that eventually correct pathogenic variants in patients.

Moreover, the principle of generating clean backgrounds for disease modeling fosters confidence that the observed phenotypes reflect the intended single mutation or cluster of mutations, rather than confounding off-target effects in the nuclear genome. As we scaled from single-blastocyst analyses to whole-litter screens and then entire F0 and F1 cohorts, we saw no evidence of spurious large-scale nuclear edits or integration events, which historically have bedeviled certain gene-editing tools. This is crucial not only for basic science but also for any translational endeavor where specificity is key to meeting regulatory standards for safety.

Another intriguing aspect of the new mitochondrially targeted editors is their compatibility with circular RNA (circRNA). CircRNA vectors are more stable than linear mRNAs, conferring prolonged expression. This was reflected in the heightened editing efficiencies in mouse embryos injected with circRNA constructs, where sustained editor expression presumably facilitated more comprehensive editing of mtDNA. The successful translation of circRNA in mitochondria-targeted editing underscores the broader potential of circular RNA technologies across various domains of gene therapy, from disease modeling in preclinical species to future therapeutic interventions in humans.

In sum, the research on mitoBEs v2 ushers in a new era of mitochondrial disease modeling, bridging the gap between theoretical constructs of disease-causing mutations and fully realized mouse lines that recapitulate human syndromes. The capacity to achieve editing efficiencies of up to 82% in F0 mice, combined with the successful demonstration of maternal inheritance and phenotypic manifestations closely matching known human conditions, is a definitive testament to the power of these refined genome-editing tools. These mouse models have already yielded insights into how pathogenic mtDNA influences embryonic and tissue-specific viability, how the mutation load can shift dramatically from mother to offspring, and how single-point changes in critical mitochondrial proteins directly provoke the cardinal features of Leigh syndrome or LHON. For the research community, the newly minted mouse lines that mimic other mitochondrial disorders—be it through tRNA mutations or protein-coding gene alterations—offer an expansive toolkit to elucidate unknown mechanisms and test novel therapeutic strategies.

The quest for single-base editing in mitochondrial DNA, once limited by a dearth of tools and hampered by the intricacy of delivering effectors to mitochondria, is now coming to fruition. The lessons learned from the iterative optimization of mitoBEs v2—especially the efforts to minimize RNA off-target edits and reduce promiscuous deaminase activity—will undoubtedly inform future developments, perhaps enabling the next generation of mitochondrially targeted editors to be even safer and more precise. Ultimately, the synergy between robust mitochondrial base editing and advanced in vivo delivery systems could herald genuine clinical interventions for a wide spectrum of mitochondrial disorders, enabling the possibility that familial burdens associated with debilitating mutations can be alleviated by precise reprogramming of the mitochondrial genome. Far from being a mere technical feat, these breakthroughs embody a promising leap toward conquering inherited metabolic conditions that have long stood as insurmountable clinical challenges.

Subject of Research: Mitochondrial genome base editing to create mouse models of human mitochondrial diseases

Article Title : Precise modelling of mitochondrial diseases using optimized mitoBEs

News Publication Date : 22 January 2025

Keywords : Mitochondrial diseases, base editing, mitoBEs v2, genome editing, animal models, TALE-fused deaminases, off-target effects, maternal inheritance, disease phenotypes, Leigh syndrome, LHON, circRNA technology