Статті в журналах: "MtDNA editing"

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Jo, Areum, Sangwoo Ham, Gum Hwa Lee, Yun-Il Lee, SangSeong Kim, Yun-Song Lee, Joo-Ho Shin, and Yunjong Lee. "Efficient Mitochondrial Genome Editing by CRISPR/Cas9." BioMed Research International 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/305716.

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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing.

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Best, Corinne, Ron Mizrahi, and Oren Ostersetzer-Biran. "Why so Complex? The Intricacy of Genome Structure and Gene Expression, Associated with Angiosperm Mitochondria, May Relate to the Regulation of Embryo Quiescence or Dormancy—Intrinsic Blocks to Early Plant Life." Plants 9, no. 5 (May 8, 2020): 598. http://dx.doi.org/10.3390/plants9050598.

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Mitochondria play key roles in cellular-energy metabolism and are vital for plant-life, such as for successful germination and early-seedling establishment. Most mitochondria contain their own genetic system (mtDNA, mitogenome), with an intrinsic protein-synthesis machinery. Although the challenges of maintaining prokaryotic-type structures and functions are common to Eukarya, land plants possess some of the most complex organelle composition of all known organisms. Angiosperms mtDNAs are characteristically the largest and least gene-dense among the eukaryotes. They often contain highly-variable intergenic regions of endogenous or foreign origins and undergo frequent recombination events, which result in different mtDNA configurations, even between closely-related species. The expression of the mitogenome in angiosperms involves extensive mtRNA processing steps, including numerous editing and splicing events. Why do land-plant’s mitochondria have to be so complex? The answer to this remains a matter of speculation. We propose that this complexity may have arisen throughout the terrestrialization of plants, as a means to control embryonic mitochondrial functions —a critical adaptive trait to optimize seed germination. The unique characteristics of plant mtDNA may play pivotal roles in the nuclear-regulation of organellar biogenesis and metabolism, possibly to control embryos quiescence or dormancy, essential determinants for the establishment of viable plantlets that can survive post-germination.

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Yamada, Mitsutoshi, Kazuhiro Akashi, Reina Ooka, Kenji Miyado, and Hidenori Akutsu. "Mitochondrial Genetic Drift after Nuclear Transfer in Oocytes." International Journal of Molecular Sciences 21, no. 16 (August 16, 2020): 5880. http://dx.doi.org/10.3390/ijms21165880.

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Mitochondria are energy-producing intracellular organelles containing their own genetic material in the form of mitochondrial DNA (mtDNA), which codes for proteins and RNAs essential for mitochondrial function. Some mtDNA mutations can cause mitochondria-related diseases. Mitochondrial diseases are a heterogeneous group of inherited disorders with no cure, in which mutated mtDNA is passed from mothers to offspring via maternal egg cytoplasm. Mitochondrial replacement (MR) is a genome transfer technology in which mtDNA carrying disease-related mutations is replaced by presumably disease-free mtDNA. This therapy aims at preventing the transmission of known disease-causing mitochondria to the next generation. Here, a proof of concept for the specific removal or editing of mtDNA disease-related mutations by genome editing is introduced. Although the amount of mtDNA carryover introduced into human oocytes during nuclear transfer is low, the safety of mtDNA heteroplasmy remains a concern. This is particularly true regarding donor-recipient mtDNA mismatch (mtDNA–mtDNA), mtDNA-nuclear DNA (nDNA) mismatch caused by mixing recipient nDNA with donor mtDNA, and mtDNA replicative segregation. These conditions can lead to mtDNA genetic drift and reversion to the original genotype. In this review, we address the current state of knowledge regarding nuclear transplantation for preventing the inheritance of mitochondrial diseases.

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de Oliveira, Vanessa Cristina, Kelly Cristine Santos Roballo, Clésio Gomes Mariano Junior, Sarah Ingrid Pinto Santos, Fabiana Fernandes Bressan, Marcos Roberto Chiaratti, Elena J. Tucker, Erica E. Davis, Jean-Paul Concordet, and Carlos Eduardo Ambrósio. "HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation." Life 12, no. 1 (December 24, 2021): 22. http://dx.doi.org/10.3390/life12010022.

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The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67–96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.

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5

Zheng, Yang. "Application and Challenge of CRISPR System to Mitochondrial Genetic Disorders." Highlights in Science, Engineering and Technology 91 (April 15, 2024): 289–98. http://dx.doi.org/10.54097/n26n2410.

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A distinct class of genetic illnesses known as mitochondrial genetic disorders is brought on by genetic mutations in the mitochondrial DNA. The lowest prevalence of mtDNA mutations is 1 in 5,000, leading to mitochondrial genetic disorders for which there are yet no economical diagnostic methods or therapeutic interventions. The CRISPR system is an immune defense system of prokaryotes that can recognize and cut foreign DNA, inhibit the expression of foreign genes, and fend off viral interference. It is because of its precise targeting ability that the CRISPR/Cas system has been transformed into a highly effective gene editing technique. Future treatments for mitochondrial illnesses may benefit from the CRISPR system, as well as the successful development of mitochondrial gene editing instruments. By referring to both domestic and international literature, this paper introduces Crispr/Cas technology and mitochondrial genes, summarizes specific cases of CRISPR system applied to mitochondrial gene, and focuses on the technical limitations of Crispr system for mtDNA modification. There is also discussion of the application prospect of CRISPR system in mtDNA modification. At present, it is found that the main challenge impeding the advancement of mtDNA editing technology within the CRISPR system is the technology for gRNA to gain entry into the mitochondria.

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Klucnika, Anna, and Hansong Ma. "Mapping and editing animal mitochondrial genomes: can we overcome the challenges?" Philosophical Transactions of the Royal Society B: Biological Sciences 375, no. 1790 (December 2, 2019): 20190187. http://dx.doi.org/10.1098/rstb.2019.0187.

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The animal mitochondrial genome, although small, can have a big impact on health and disease. Non-pathogenic sequence variation among mitochondrial DNA (mtDNA) haplotypes influences traits including fertility, healthspan and lifespan, whereas pathogenic mutations are linked to incurable mitochondrial diseases and other complex conditions like ageing, diabetes, cancer and neurodegeneration. However, we know very little about how mtDNA genetic variation contributes to phenotypic differences. Infrequent recombination, the multicopy nature and nucleic acid-impenetrable membranes present significant challenges that hamper our ability to precisely map mtDNA variants responsible for traits, and to genetically modify mtDNA so that we can isolate specific mutants and characterize their biochemical and physiological consequences. Here, we summarize the past struggles and efforts in developing systems to map and edit mtDNA. We also assess the future of performing forward and reverse genetic studies on animal mitochondrial genomes. This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.

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Herbert, Mary. "Genome Editing Tools to Increase the Efficacy of Mitochondrial Donation." Fertility & Reproduction 05, no. 04 (December 2023): 259. http://dx.doi.org/10.1142/s2661318223740730.

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Mitochondrial donation (MD) aims to prevent transmission of mtDNA disease by transplanting the nuclear genome from an affected woman’s egg to an enucleated egg from an unaffected donor. The nuclear DNA is transplanted in a karyoplast, which upon fusion with the enucleated donor egg, introduces a small amount of perinuclear mitochondria resulting in heteroplasmy for maternal mtDNA. Under optimal conditions, this accounts for <2% of the mtDNA content of MD embryos. Despite this, [Formula: see text]20% of embryonic stem (ES) cell lines derived from MD embryos exhibit complete reversion to the maternal mitochondrial genome and elevated levels (40–60%) have recently been reported in a baby born following MD treatment for infertility. Thus, currently available MR treatments are regarded as risk reduction, rather than prevention strategies. A major focus of our ongoing research is to bridge this gap by developing techniques to minimise maternal mtDNA in MD embryos. I will present our recent findings from these investigations.

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Zhong, Gang, Henning Madry, and Magali Cucchiarini. "Mitochondrial Genome Editing to Treat Human Osteoarthritis—A Narrative Review." International Journal of Molecular Sciences 23, no. 3 (January 27, 2022): 1467. http://dx.doi.org/10.3390/ijms23031467.

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Osteoarthritis (OA) is a severe, common chronic orthopaedic disorder characterised by a degradation of the articular cartilage with an incidence that increases over years. Despite the availability of various clinical options, none can stop the irreversible progression of the disease to definitely cure OA. Various mutations have been evidenced in the mitochondrial DNA (mtDNA) of cartilage cells (chondrocytes) in OA, leading to a dysfunction of the mitochondrial oxidative phosphorylation processes that significantly contributes to OA cartilage degeneration. The mitochondrial genome, therefore, represents a central, attractive target for therapy in OA, especially using genome editing procedures. In this narrative review article, we present and discuss the current advances and breakthroughs in mitochondrial genome editing as a potential, novel treatment to overcome mtDNA-related disorders such as OA. While still in its infancy and despite a number of challenges that need to be addressed (barriers to effective and site-specific mtDNA editing and repair), such a strategy has strong value to treat human OA in the future, especially using the groundbreaking clustered regularly interspaced short palindromic repeats (CRIPSR)/CRISPR-associated 9 (CRISPR/Cas9) technology and mitochondrial transplantation approaches.

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Moraes, Carlos T. "Tools for editing the mammalian mitochondrial genome." Human Molecular Genetics 33, R1 (May 22, 2024): R92—R99. http://dx.doi.org/10.1093/hmg/ddae037.

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Abstract 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.

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Moreira, Jesse D., Deepa M. Gopal, Darrell N. Kotton, and Jessica L. Fetterman. "Gaining Insight into Mitochondrial Genetic Variation and Downstream Pathophysiology: What Can i(PSCs) Do?" Genes 12, no. 11 (October 22, 2021): 1668. http://dx.doi.org/10.3390/genes12111668.

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Mitochondria are specialized organelles involved in energy production that have retained their own genome throughout evolutionary history. The mitochondrial genome (mtDNA) is maternally inherited and requires coordinated regulation with nuclear genes to produce functional enzyme complexes that drive energy production. Each mitochondrion contains 5–10 copies of mtDNA and consequently, each cell has several hundreds to thousands of mtDNAs. Due to the presence of multiple copies of mtDNA in a mitochondrion, mtDNAs with different variants may co-exist, a condition called heteroplasmy. Heteroplasmic variants can be clonally expanded, even in post-mitotic cells, as replication of mtDNA is not tied to the cell-division cycle. Heteroplasmic variants can also segregate during germ cell formation, underlying the inheritance of some mitochondrial mutations. Moreover, the uneven segregation of heteroplasmic variants is thought to underlie the heterogeneity of mitochondrial variation across adult tissues and resultant differences in the clinical presentation of mitochondrial disease. Until recently, however, the mechanisms mediating the relation between mitochondrial genetic variation and disease remained a mystery, largely due to difficulties in modeling human mitochondrial genetic variation and diseases. The advent of induced pluripotent stem cells (iPSCs) and targeted gene editing of the nuclear, and more recently mitochondrial, genomes now provides the ability to dissect how genetic variation in mitochondrial genes alter cellular function across a variety of human tissue types. This review will examine the origins of mitochondrial heteroplasmic variation and propagation, and the tools used to model mitochondrial genetic diseases. Additionally, we discuss how iPSC technologies represent an opportunity to advance our understanding of human mitochondrial genetics in disease.

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Hammar, Freya, and Dennis L. Miller. "Genetic Diversity in the mtDNA of Physarum polycephalum." Genes 14, no. 3 (March 2, 2023): 628. http://dx.doi.org/10.3390/genes14030628.

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The mtDNA of the myxomycete Physarum polycephalum can contain as many as 81 genes. These genes can be grouped in three different categories. The first category includes 46 genes that are classically found on the mtDNA of many organisms. However, 43 of these genes are cryptogenes that require a unique type of RNA editing (MICOTREM). A second category of gene is putative protein-coding genes represented by 26 significant open reading frames. However, these genes do not appear to be transcribed during the growth of the plasmodium and are currently unassigned since they do not have any apparent similarity to other classical mitochondrial protein-coding genes. The third category of gene is found in the mtDNA of some strains of P. polycephalum. These genes derive from a linear mitochondrial plasmid with nine significant, but unassigned, open reading frames which can integrate into the mitochondrial DNA by recombination. Here, we review the mechanism and evolution of the RNA editing necessary for cryptogene expression, discuss possible origins for the 26 unassigned open reading frames based on tentative identification of their protein product, and discuss the implications to mtDNA structure and replication of the integration of the linear mitochondrial plasmid.

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Rai, Pavandeep K., Lyndsey Craven, Kurt Hoogewijs, Oliver M. Russell, and Robert N. Lightowlers. "Advances in methods for reducing mitochondrial DNA disease by replacing or manipulating the mitochondrial genome." Essays in Biochemistry 62, no. 3 (June 27, 2018): 455–65. http://dx.doi.org/10.1042/ebc20170113.

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Mitochondrial DNA (mtDNA) is a multi-copy genome whose cell copy number varies depending on tissue type. Mutations in mtDNA can cause a wide spectrum of diseases. Mutated mtDNA is often found as a subset of the total mtDNA population in a cell or tissue, a situation known as heteroplasmy. As mitochondrial dysfunction only presents after a certain level of heteroplasmy has been acquired, ways to artificially reduce or replace the mutated species have been attempted. This review addresses recent approaches and advances in this field, focusing on the prevention of pathogenic mtDNA transfer via mitochondrial donation techniques such as maternal spindle transfer and pronuclear transfer in which mutated mtDNA in the oocyte or fertilized embryo is substituted with normal copies of the mitochondrial genome. This review also discusses the molecular targeting and cleavage of pathogenic mtDNA to shift heteroplasmy using antigenomic therapy and genome engineering techniques including Zinc-finger nucleases and transcription activator-like effector nucleases. Finally, it considers CRISPR technology and the unique difficulties that mitochondrial genome editing presents.

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Kargaran, Parisa K., Jared M. Evans, Sara E. Bodbin, James G. W. Smith, Timothy J. Nelson, Chris Denning, and Diogo Mosqueira. "Mitochondrial DNA: Hotspot for Potential Gene Modifiers Regulating Hypertrophic Cardiomyopathy." Journal of Clinical Medicine 9, no. 8 (July 23, 2020): 2349. http://dx.doi.org/10.3390/jcm9082349.

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Hypertrophic cardiomyopathy (HCM) is a prevalent and untreatable cardiovascular disease with a highly complex clinical and genetic causation. HCM patients bearing similar sarcomeric mutations display variable clinical outcomes, implying the involvement of gene modifiers that regulate disease progression. As individuals exhibiting mutations in mitochondrial DNA (mtDNA) present cardiac phenotypes, the mitochondrial genome is a promising candidate to harbor gene modifiers of HCM. Herein, we sequenced the mtDNA of isogenic pluripotent stem cell-cardiomyocyte models of HCM focusing on two sarcomeric mutations. This approach was extended to unrelated patient families totaling 52 cell lines. By correlating cellular and clinical phenotypes with mtDNA sequencing, potentially HCM-protective or -aggravator mtDNA variants were identified. These novel mutations were mostly located in the non-coding control region of the mtDNA and did not overlap with those of other mitochondrial diseases. Analysis of unrelated patients highlighted family-specific mtDNA variants, while others were common in particular population haplogroups. Further validation of mtDNA variants as gene modifiers is warranted but limited by the technically challenging methods of editing the mitochondrial genome. Future molecular characterization of these mtDNA variants in the context of HCM may identify novel treatments and facilitate genetic screening in cardiomyopathy patients towards more efficient treatment options.

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Kozhukhar, Natalya, Domenico Spadafora, Yelitza A. R. Rodriguez, and Mikhail F. Alexeyev. "A Method for In Situ Reverse Genetic Analysis of Proteins Involved mtDNA Replication." Cells 11, no. 14 (July 11, 2022): 2168. http://dx.doi.org/10.3390/cells11142168.

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The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial transcription, and respiratory chain function. Finally, we present evidence that the HMG2 domain plays the leading role in TFAM species-specificity, thus indicating a potential pathway for TFAM-mtDNA evolutionary co-adaptations.

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Forner, Joachim, Dennis Kleinschmidt, Etienne H. Meyer, Axel Fischer, Robert Morbitzer, Thomas Lahaye, Mark A. Schöttler, and Ralph Bock. "Targeted introduction of heritable point mutations into the plant mitochondrial genome." Nature Plants 8, no. 3 (March 2022): 245–56. http://dx.doi.org/10.1038/s41477-022-01108-y.

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AbstractThe development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.

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Hattori, Nobuaki, Kazuaki Kitagawa, Shigeo Takumi, and Chiharu Nakamura. "Mitochondrial DNA Heteroplasmy in Wheat, Aegilops and Their Nucleus-Cytoplasm Hybrids." Genetics 160, no. 4 (April 1, 2002): 1619–30. http://dx.doi.org/10.1093/genetics/160.4.1619.

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Abstract A mitochondrial (mt) transcriptional unit, nad3-orf156, was studied in the nucleus-cytoplasm hybrids of wheat with D/D2 plasmons from Aegilops species and their parental lines. A comparative RFLP analysis and sequencing of the random PCR clones revealed the presence of seven sequence types and their polymorphic sites were mapped. All the hybrids possessed the paternal copies besides the maternal copies. More paternal copies were present in the D2 plasmon hybrids, whereas more maternal copies were present in the D plasmon hybrids. Two major copies were present with different stoichiometries in the maternal Aegilops parents. However, only a major D plasmon copy was detected in the hybrids, irrespective of their plasmon types. The hexaploid wheat parent (AABBDD genome) possessed the major D plasmon copy in ~5% stoichiometry, while no D plasmon-homologous copies were detected in the tetraploid wheat parent (AABB genome). The results suggest that the observed mtDNA heteroplasmy is due to paternal contribution of mtDNA. The different copy stoichiometry suggests differential amplification of the heteroplasmic copies among the hybrids and the parental lines. All editing sites and their editing frequencies were conserved among the lines, and only the maternal pattern of editing occurred in the hybrids.

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Zekonyte, U., S. R. Bacman, and C. T. Moraes. "DNA‐editing enzymes as potential treatments for heteroplasmic mtDNA diseases." Journal of Internal Medicine 287, no. 6 (April 27, 2020): 685–97. http://dx.doi.org/10.1111/joim.13055.

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18

Varré, D’Agostino, Touzet, Gallina, Tamburino, Cantarella, Ubrig, et al. "Complete Sequence, Multichromosomal Architecture and Transcriptome Analysis of the Solanum tuberosum Mitochondrial Genome." International Journal of Molecular Sciences 20, no. 19 (September 26, 2019): 4788. http://dx.doi.org/10.3390/ijms20194788.

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Mitochondrial genomes (mitogenomes) in higher plants can induce cytoplasmic male sterility and be somehow involved in nuclear-cytoplasmic interactions affecting plant growth and agronomic performance. They are larger and more complex than in other eukaryotes, due to their recombinogenic nature. For most plants, the mitochondrial DNA (mtDNA) can be represented as a single circular chromosome, the so-called master molecule, which includes repeated sequences that recombine frequently, generating sub-genomic molecules in various proportions. Based on the relevance of the potato crop worldwide, herewith we report the complete mtDNA sequence of two S. tuberosum cultivars, namely Cicero and Désirée, and a comprehensive study of its expression, based on high-coverage RNA sequencing data. We found that the potato mitogenome has a multi-partite architecture, divided in at least three independent molecules that according to our data should behave as autonomous chromosomes. Inter-cultivar variability was null, while comparative analyses with other species of the Solanaceae family allowed the investigation of the evolutionary history of their mitogenomes. The RNA-seq data revealed peculiarities in transcriptional and post-transcriptional processing of mRNAs. These included co-transcription of genes with open reading frames that are probably expressed, methylation of an rRNA at a position that should impact translation efficiency and extensive RNA editing, with a high proportion of partial editing implying frequent mis-targeting by the editing machinery.

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Camacho, Esther, Alberto Rastrojo, África Sanchiz, Sandra González-de la Fuente, Begoña Aguado, and Jose M. Requena. "Leishmania Mitochondrial Genomes: Maxicircle Structure and Heterogeneity of Minicircles." Genes 10, no. 10 (September 26, 2019): 758. http://dx.doi.org/10.3390/genes10100758.

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The mitochondrial DNA (mtDNA), which is present in almost all eukaryotic organisms, is a useful marker for phylogenetic studies due to its relative high conservation and its inheritance manner. In Leishmania and other trypanosomatids, the mtDNA (also referred to as kinetoplast DNA or kDNA) is composed of thousands of minicircles and a few maxicircles, catenated together into a complex network. Maxicircles are functionally similar to other eukaryotic mtDNAs, whereas minicircles are involved in RNA editing of some maxicircle-encoded transcripts. Next-generation sequencing (NGS) is increasingly used for assembling nuclear genomes and, currently, a large number of genomic sequences are available. However, most of the time, the mitochondrial genome is ignored in the genome assembly processes. The aim of this study was to develop a pipeline to assemble Leishmania minicircles and maxicircle DNA molecules, exploiting the raw data generated in the NGS projects. As a result, the maxicircle molecules and the plethora of minicircle classes for Leishmania major, Leishmania infantum and Leishmania braziliensis have been characterized. We have observed that whereas the heterogeneity of minicircle sequences existing in a single cell hampers their use for Leishmania typing and classification, maxicircles emerge as an extremely robust genetic marker for taxonomic studies within the clade of kinetoplastids.

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Marande, William, Julius Lukeš, and Gertraud Burger. "Unique Mitochondrial Genome Structure in Diplonemids, the Sister Group of Kinetoplastids." Eukaryotic Cell 4, no. 6 (June 2005): 1137–46. http://dx.doi.org/10.1128/ec.4.6.1137-1146.2005.

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ABSTRACT Kinetoplastid flagellates are characterized by uniquely massed mitochondrial DNAs (mtDNAs), the kinetoplasts. Kinetoplastids of the trypanosomatid group possess two types of mtDNA molecules: maxicircles bearing protein and mitoribosomal genes and minicircles specifying guide RNAs, which mediate uridine insertion/deletion RNA editing. These circles are interlocked with one another to form dense networks. Whether these peculiar mtDNA features are restricted to kinetoplastids or prevail throughout Euglenozoa (euglenids, diplonemids, and kinetoplastids) is unknown. Here, we describe the mitochondrial genome and the mitochondrial ultrastructure of Diplonema papillatum, a member of the diplonemid flagellates, the sister group of kinetoplastids. Fluorescence and electron microscopy show a single mitochondrion per cell with an ultrastructure atypical for Euglenozoa. In addition, DNA is evenly distributed throughout the organelle rather than compacted. Molecular and electron microscopy studies distinguish numerous 6- and 7-kbp-sized mitochondrial chromosomes of monomeric circular topology and relaxed conformation in vivo. Remarkably, the cox1 gene (and probably other mitochondrial genes) is fragmented, with separate gene pieces encoded on different chromosomes. Generation of the contiguous cox1 mRNA requires trans-splicing, the precise mechanism of which remains to be determined. Taken together, the mitochondrial gene/genome structure of Diplonema is not only different from that of kinetoplastids but unique among eukaryotes as a whole.

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Gammage, Payam A., Carlo Viscomi, Marie-Lune Simard, Ana S. H. Costa, Edoardo Gaude, Christopher A. Powell, Lindsey Van Haute, et al. "Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo." Nature Medicine 24, no. 11 (September 24, 2018): 1691–95. http://dx.doi.org/10.1038/s41591-018-0165-9.

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Saravanan, Sanjana, Caitlin J. Lewis, Bhavna Dixit, Matthew S. O’Connor, Alexandra Stolzing, and Amutha Boominathan. "The Mitochondrial Genome in Aging and Disease and the Future of Mitochondrial Therapeutics." Biomedicines 10, no. 2 (February 18, 2022): 490. http://dx.doi.org/10.3390/biomedicines10020490.

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Mitochondria are intracellular organelles that utilize nutrients to generate energy in the form of ATP by oxidative phosphorylation. Mitochondrial DNA (mtDNA) in humans is a 16,569 base pair double-stranded circular DNA that encodes for 13 vital proteins of the electron transport chain. Our understanding of the mitochondrial genome’s transcription, translation, and maintenance is still emerging, and human pathologies caused by mtDNA dysfunction are widely observed. Additionally, a correlation between declining mitochondrial DNA quality and copy number with organelle dysfunction in aging is well-documented in the literature. Despite tremendous advancements in nuclear gene-editing technologies and their value in translational avenues, our ability to edit mitochondrial DNA is still limited. In this review, we discuss the current therapeutic landscape in addressing the various pathologies that result from mtDNA mutations. We further evaluate existing gene therapy efforts, particularly allotopic expression and its potential to become an indispensable tool for restoring mitochondrial health in disease and aging.

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23

Ota, Azusa, Takaya Ishihara, and Naotada Ishihara. "Mitochondrial nucleoid morphology and respiratory function are altered in Drp1-deficient HeLa cells." Journal of Biochemistry 167, no. 3 (December 24, 2019): 287–94. http://dx.doi.org/10.1093/jb/mvz112.

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Abstract Mitochondria are dynamic organelles that frequently divide and fuse with each other. The dynamin-related GTPase protein Drp1 has a key role in mitochondrial fission. To analyse the physiological roles of Drp1 in cultured human cells, we analysed Drp1-deficient HeLa cells established by genome editing using CRISPR/Cas9. Under fluorescent microscopy, not only mitochondria were elongated but their DNA (mtDNA) nucleoids were extremely enlarged in bulb-like mitochondrial structures (‘mito-bulbs’) in the Drp1-deficient HeLa cells. We further found that respiratory activity, as measured by oxygen consumption rates, was severely repressed in Drp1-deficient HeLa cells and that this was reversible by the co-repression of mitochondrial fusion factors. Although mtDNA copy number was not affected, several respiratory subunits were repressed in Drp1-deficient HeLa cells. These results suggest that mitochondrial fission is required for the maintenance of active respiratory activity and the morphology of mtDNA nucleoids in human cells.

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Anderson, Andrew P., Xuemei Luo, William Russell, and Y. Whitney Yin. "Oxidative damage diminishes mitochondrial DNA polymerase replication fidelity." Nucleic Acids Research 48, no. 2 (December 4, 2019): 817–29. http://dx.doi.org/10.1093/nar/gkz1018.

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Abstract Mitochondrial DNA (mtDNA) resides in a high ROS environment and suffers more mutations than its nuclear counterpart. Increasing evidence suggests that mtDNA mutations are not the results of direct oxidative damage, rather are caused, at least in part, by DNA replication errors. To understand how the mtDNA replicase, Pol γ, can give rise to elevated mutations, we studied the effect of oxidation of Pol γ on replication errors. Pol γ is a high fidelity polymerase with polymerase (pol) and proofreading exonuclease (exo) activities. We show that Pol γ exo domain is far more sensitive to oxidation than pol; under oxidative conditions, exonuclease activity therefore declines more rapidly than polymerase. The oxidized Pol γ becomes editing-deficient, displaying a 20-fold elevated mutations than the unoxidized enzyme. Mass spectrometry analysis reveals that Pol γ exo domain is a hotspot for oxidation. The oxidized exo residues increase the net negative charge around the active site that should reduce the affinity to mismatched primer/template DNA. Our results suggest that the oxidative stress induced high mutation frequency on mtDNA can be indirectly caused by oxidation of the mitochondrial replicase.

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25

Liu, Yu, Yuejia Huang, Chong Xu, Peng An, Yongting Luo, Lei Jiao, Junjie Luo, and Yongzhi Li. "Mitochondrial Dysfunction and Therapeutic Perspectives in Cardiovascular Diseases." International Journal of Molecular Sciences 23, no. 24 (December 16, 2022): 16053. http://dx.doi.org/10.3390/ijms232416053.

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High mortality rates due to cardiovascular diseases (CVDs) have attracted worldwide attention. It has been reported that mitochondrial dysfunction is one of the most important mechanisms affecting the pathogenesis of CVDs. Mitochondrial DNA (mtDNA) mutations may result in impaired oxidative phosphorylation (OXPHOS), abnormal respiratory chains, and ATP production. In dysfunctional mitochondria, the electron transport chain (ETC) is uncoupled and the energy supply is reduced, while reactive oxygen species (ROS) production is increased. Here, we discussed and analyzed the relationship between mtDNA mutations, impaired mitophagy, decreased OXPHOS, elevated ROS, and CVDs from the perspective of mitochondrial dysfunction. Furthermore, we explored current potential therapeutic strategies for CVDs by eliminating mtDNA mutations (e.g., mtDNA editing and mitochondrial replacement), enhancing mitophagy, improving OXPHOS capacity (e.g., supplement with NAD+, nicotinamide riboside (NR), nicotinamide mononucleotide (NMN), and nano-drug delivery), and reducing ROS (e.g., supplement with Coenzyme Q10 and other antioxidants), and dissected their respective advantages and limitations. In fact, some therapeutic strategies are still a long way from achieving safe and effective clinical treatment. Although establishing effective and safe therapeutic strategies for CVDs remains challenging, starting from a mitochondrial perspective holds bright prospects.

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Kar, Bibekananda, Santiago R. Castillo, Ankit Sabharwal, Karl J. Clark, and Stephen C. Ekker. "Mitochondrial Base Editing: Recent Advances towards Therapeutic Opportunities." International Journal of Molecular Sciences 24, no. 6 (March 18, 2023): 5798. http://dx.doi.org/10.3390/ijms24065798.

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Mitochondria are critical organelles that form networks within our cells, generate energy dynamically, contribute to diverse cell and organ function, and produce a variety of critical signaling molecules, such as cortisol. This intracellular microbiome can differ between cells, tissues, and organs. Mitochondria can change with disease, age, and in response to the environment. Single nucleotide variants in the circular genomes of human mitochondrial DNA are associated with many different life-threatening diseases. Mitochondrial DNA base editing tools have established novel disease models and represent a new possibility toward personalized gene therapies for the treatment of mtDNA-based disorders.

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27

Zein, Muhammad Ihda Hamlu Liwaissunati, Ari Hardianto, Irkham Irkham, and Yeni Wahyuni Hartati. "Identification of CRISPR/Cas12a (Cpf1) guideRNA Sequence Targeting the Mitochondrial DNA D-loop Region in Wild Pig (Sus scrofa) Through Homology Difference and Mismatch Analysis." Trends in Sciences 21, no. 5 (March 20, 2024): 7603. http://dx.doi.org/10.48048/tis.2024.7603.

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CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) or CRISPR-associated (Cas) System has become a major gene editing tool. Gene editing with CRISPR requires the Cas protein and the corresponding guide RNA (gRNA). However, low cleavage efficiency and off-target effects can impede the application of the CRISPR/Cas system. Therefore, the determination of specific gRNAs is essential. In biosensor applications, CRISPR/Cas12a can enhance specificity and sensitivity in identifying target genes due to the trans-cleavage activity of Cas12a (Cpf1). The mtDNA D-loop sequence is the most variable part of mtDNA, making it suitable for distinguishing between species. Consequently, the objective of this study was to determine the gRNA sequence of the D-loop of wild pig mtDNA in silico. Candidate gRNAs were predicted using the Benchling application with the assistance of the GenBank database. The gRNA candidates were subsequently subjected to a homology difference analysis using BLAST nucleotide and a mismatch test using Jalview. Among several candidates, candidate 1 was selected as the best option, with an Off-target value of 99.8. The homology difference analysis against competitors and the mismatch test against the Sus genus resulted in high E-values and high percentage values, respectively. This suggests that the candidate will not recognize other species but can detect members of the Sus scrofa species. These gRNA candidates can be selectively and sensitively applied to biosensors for the detection of meat adulteration. HIGHLIGHTS The D-loop exhibits high variability, make it useful for distinguishing between species gRNA as an enabler of CRISPR/Cas12a to identify the target and initiate cleavage activity The candidate 1 was selected with the sequence 5'-GAT TGT CGT GCC GGA TCA TGA GTT-3' The application of this strategy is aimed at determining the halal status of food products The application will encompass both quantitative and qualitative aspects GRAPHICAL ABSTRACT

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28

Klopstock, Thomas, Leopold H. Zeng, and Claudia Priglinger. "Leber’s hereditary optic neuropathy – current status of idebenone and gene replacement therapies." Medizinische Genetik 37, no. 1 (February 6, 2025): 57–63. https://doi.org/10.1515/medgen-2024-2066.

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Abstract Leber’s hereditary optic neuropathy (LHON) is the most common mitochondrial disease, and was the first to be linked to mitochondrial DNA (mtDNA) variations. Recently, autosomal recessive forms of LHON were described in addition to the classical mtDNA-associated forms. Clinically, LHON manifests with subacute and painless loss of central visual acuity, in most cases starting unilaterally, and involving the second eye a few weeks later. Almost all LHON cases are caused by pathogenic variants in genes that code for proteins relevant for function of Complex I of the respiratory chain. The Complex I dysfunction in LHON leads to decreased ATP synthesis and to increased production of reactive oxygen species which ultimately initiates dysfunction and apoptosis of retinal ganglion cells and their axons, the optic nerve. Idebenone, a synthetic CoQ derivative, is a potent intramitochondrial antioxidant and can shuttle electrons directly to complex III of the respiratory chain, thereby bypassing complex I deficiency. On the basis of several clinical trials, it has been approved as a treatment for LHON in 2015 (in the EU). In addition, direct intravitreal gene replacement therapy is being investigated, with several late-stage clinical trials already completed. In the future, gene editing of mtDNA variants may also become a therapeutic option.

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Ward, Grace A., Kathy McGraw, Amy F. McLemore, Nghi B. Lam, Hsin-An Hou, Benjamin S. Meyer, and Alan F. List. "Oxidized Mitochondrial DNA Engages TLR9 to Activate the NLRP3 Inflammasome in Myelodysplastic Syndromes." Blood 134, Supplement_1 (November 13, 2019): 774. http://dx.doi.org/10.1182/blood-2019-122358.

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Myelodysplastic Syndromes (MDS) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, Nlrp3 inflammasome (IFM) driven pyroptotic cell death and the induction of interferon-stimulated genes (ISG). Toll-like receptor 9 (TLR9) is an endosomal, DNA sensing pattern recognition receptor that primes and activates the IFM and ISG response through myddosome signaling upon engagement by hypomethylated, CpG-rich DNA. Oxidized newly synthesized mitochondrial DNA (ox-mtDNA) is released into the cytosol upon TLR/IL-1R activation to trigger Nlrp3 IFM activation. Upon lytic pyroptotic cell death, however, ox-mtDNA is released into the extracellular space. We previously reported that concentrations of ox-mtDNA, a native TLR9 ligand, are profoundly increased in MDS patient plasma compared to age-matched controls and other hematologic malignancies (Ward G, et. al. ASH 2018). The aim of this investigation was to determine if ox-mtDNA acts as a danger associated molecular pattern (DAMP) to propagate the inflammatory response and IFM activation in neighboring cells through TLR9. We have shown that MDS hematopoietic stem and progenitor cells (HSPC) redistribute TLR9 to display cell surface TLR9 expression. We hypothesized that this increased surface expression is induced in response to ox-mtDNA in the BM plasma. To test this, SKM1 and U937 cells were incubated for 2 hours with 50ng/mL ox-mtDNA (ND1 gene, unmethylated, amplified with oxidized guanosine) and TLR9 expression was assessed by flow cytometry (FC). Following incubation, both cell lines significantly increased TLR9 surface expression (n=3, p<0.03).We next confirmed that TLR9 interacts with ox-mtDNA by immunofluorescence (IF) microscopy in MDS samples and murine somatic gene mutation (SGM) models (Tet2-/- and Srsf2P95H). We further investigated the relationship between TLR9 and ox-mtDNA during IFM activation. Treatment of SKM1 and U937 cells with the TLR4 ligand LPS and priming agents ATP/nigericin initiates a robust increase in ox-mtDNA that co-localized intracellularly with TLR9 by IF microscopy. Furthermore, upon incubation with ox-mtDNA, ox-mtDNA bound TLR9 is internalized. Additionally, interferon regulatory factor 7 (IRF7), an ISG transcription factor induced by TLR9, translocates to the nucleus following treatment, confirming TLR9 activation in response to ox-mtDNA. We next assessed whether IFM activation by ox-mtDNA is TLR9-dependent. 50ng/mL ox-mtDNA was incubated with 3 leukemic cell lines: SKM1, U937, and THP1 that display varying levels of endogenous TLR9 expression, as well as corresponding TLR9-KO cells generated by CRISPR-Cas9 editing. We found that TLR9 was necessary for IFM initiated caspase-1 cleavage in response to ox-mtDNA, thereby demonstrating the specificity of ox-mtDNA for the TLR9 sensor. Notably, receptor density determined response tempo. SKM1 cells, which have the highest TLR9 receptor density, responded within 1 hour, U937 cells which have an intermediate receptor density respond in 2 hours, while THP1 cells, which do not express endogenous TLR9 never responded to ox-mtDNA treatment. TLR9 knockout abrogated IFM activation in response to ox-mtDNA as demonstrated by caspase-1 glo ® assay (n=5, p<0.03). By western blot, TLR9 knockout cells no longer demonstrate phosphorylated NFk-B, cleaved caspase-1, and cleaved IL-1β in response to ox-mtDNA treatment. We conclude that MDS HSPC display functionally competent TLR9 on the plasma membrane which primes them for response to ox-mtDNA released by neighboring pyroptotic cells. Blocking TLR9 activation may prove to be a novel therapeutic strategy for MDS and suppression of inflammatory BM failure. Disclosures Hou: Celgene: Research Funding; Abbvie, Astellas, BMS, Celgene, Chugai, Daiichi Sankyo, IQVIA, Johnson & Johnson, Kirin, Merck Sharp & Dohme, Novartis, Pfizer, PharmaEssential, Roche, Takeda: Honoraria. List:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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30

Ghiselli, Fabrizio, and Liliana Milani. "Linking the mitochondrial genotype to phenotype: a complex endeavour." Philosophical Transactions of the Royal Society B: Biological Sciences 375, no. 1790 (December 2, 2019): 20190169. http://dx.doi.org/10.1098/rstb.2019.0169.

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Finding causal links between genotype and phenotype is a major issue in biology, even more in mitochondrial biology. First of all, mitochondria form complex networks, undergoing fission and fusion and we do not know how such dynamics influence the distribution of mtDNA variants across the mitochondrial network and how they affect the phenotype. Second, the non-Mendelian inheritance of mitochondrial genes can have sex-specific effects and the mechanism of mitochondrial inheritance is still poorly understood, so it is not clear how selection and/or drift act on mtDNA genetic variation in each generation. Third, we still do not know how mtDNA expression is regulated; there is growing evidence for a convoluted mechanism that includes RNA editing, mRNA stability/turnover, post-transcriptional and post-translational modifications. Fourth, mitochondrial activity differs across species as a result of several interacting processes such as drift, adaptation, genotype-by-environment interactions, mitonuclear coevolution and epistasis. This issue will cover several aspects of mitochondrial biology along the path from genotype to phenotype, and it is subdivided into four sections focusing on mitochondrial genetic variation, on the relationship among mitochondria, germ line and sex, on the role of mitochondria in adaptation and phenotypic plasticity, and on some future perspectives in mitochondrial research. This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.

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31

Picardi, Ernesto, David S. Horner, Matteo Chiara, Riccardo Schiavon, Giorgio Valle, and Graziano Pesole. "Large-scale detection and analysis of RNA editing in grape mtDNA by RNA deep-sequencing." Nucleic Acids Research 38, no. 14 (April 10, 2010): 4755–67. http://dx.doi.org/10.1093/nar/gkq202.

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32

Antón, Zuriñe, Grace Mullally, Holly C. Ford, Marc W. van der Kamp, Mark D. Szczelkun, and Jon D. Lane. "Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors." Journal of Cell Science 133, no. 18 (August 25, 2020): jcs248468. http://dx.doi.org/10.1242/jcs.248468.

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ABSTRACTCurrent methodologies for targeting the mitochondrial genome for research and/or therapy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. A molecular toolbox for CRISPR-mediated mitochondrial genome editing is desirable, as this could enable targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes. Such ‘MitoCRISPR’ systems described to date lack reproducibility and independent corroboration. We have explored the requirements for MitoCRISPR in human cells by CRISPR nuclease engineering, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of guide (g)RNA modifications for mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and effects on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Our data link mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary for MitoCRISPR.

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33

Valach, Matus, Alexandra Léveillé-Kunst, Michael W. Gray, and Gertraud Burger. "Respiratory chain Complex I of unparalleled divergence in diplonemids." Journal of Biological Chemistry 293, no. 41 (August 30, 2018): 16043–56. http://dx.doi.org/10.1074/jbc.ra118.005326.

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Mitochondrial genes of Euglenozoa (Kinetoplastida, Diplonemea, and Euglenida) are notorious for being barely recognizable, raising the question of whether such divergent genes actually code for functional proteins. Here we demonstrate the translation and identify the function of five previously unassigned y genes encoded by mitochondrial DNA (mtDNA) of diplonemids. As is the rule in diplonemid mitochondria, y genes are fragmented, with gene pieces transcribed separately and then trans-spliced to form contiguous mRNAs. Further, y transcripts undergo massive RNA editing, including uridine insertions that generate up to 16-residue-long phenylalanine tracts, a feature otherwise absent from conserved mitochondrial proteins. By protein sequence analyses, MS, and enzymatic assays in Diplonema papillatum, we show that these y genes encode the subunits Nad2, -3, -4L, -6, and -9 of the respiratory chain Complex I (CI; NADH:ubiquinone oxidoreductase). The few conserved residues of these proteins are essentially those involved in proton pumping across the inner mitochondrial membrane and in coupling ubiquinone reduction to proton pumping (Nad2, -3, -4L, and -6) and in interactions with subunits containing electron-transporting Fe-S clusters (Nad9). Thus, in diplonemids, 10 CI subunits are mtDNA-encoded. Further, MS of D. papillatum CI allowed identification of 26 conventional and 15 putative diplonemid-specific nucleus-encoded components. Most conventional accessory subunits are well-conserved but unusually long, possibly compensating for the streamlined mtDNA-encoded components and for missing, otherwise widely distributed, conventional subunits. Finally, D. papillatum CI predominantly exists as a supercomplex I:III:IV that is exceptionally stable, making this protist an organism of choice for structural studies.

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Khalfi, Pierre, Rodolphe Suspène, Kyle A. Raymond, Vincent Caval, Grégory Caignard, Noémie Berry, Valérie Thiers, et al. "Antagonism of ALAS1 by the Measles Virus V protein contributes to degradation of the mitochondrial network and promotes interferon response." PLOS Pathogens 19, no. 2 (February 21, 2023): e1011170. http://dx.doi.org/10.1371/journal.ppat.1011170.

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Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5′-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5′-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR−/− hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5’TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.

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Formosa, Luke E., Boris Reljic, Alice J. Sharpe, Daniella H. Hock, Linden Muellner-Wong, David A. Stroud, and Michael T. Ryan. "Optic atrophy–associated TMEM126A is an assembly factor for the ND4-module of mitochondrial complex I." Proceedings of the National Academy of Sciences 118, no. 17 (April 20, 2021): e2019665118. http://dx.doi.org/10.1073/pnas.2019665118.

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Mitochondrial disease is a debilitating condition with a diverse genetic etiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal-recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies, and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labeling interaction studies reveal that TMEM126A associates with the newly synthesized mitochondrial DNA (mtDNA)-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. In addition, we find that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I.

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36

Nguyen, Tan-Trung, Corinne Best, Sofia Shevtsov, Michal Zmudjak, Martine Quadrado, Ron Mizrahi, Hagit Zer, Hakim Mireau, and Oren Ostersetzer-Biran. "MISF2 Encodes an Essential Mitochondrial Splicing Cofactor Required for nad2 mRNA Processing and Embryo Development in Arabidopsis thaliana." International Journal of Molecular Sciences 23, no. 5 (February 28, 2022): 2670. http://dx.doi.org/10.3390/ijms23052670.

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Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players in plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1, and thus respiratory complex I biogenesis, are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot–dicot divergence ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.

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37

Hatzoglou, E., G. C. Rodakis, and R. Lecanidou. "Complete sequence and gene organization of the mitochondrial genome of the land snail Albinaria coerulea." Genetics 140, no. 4 (August 1, 1995): 1353–66. http://dx.doi.org/10.1093/genetics/140.4.1353.

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Abstract The complete sequence (14,130 bp) of the mitochondrial DNA (mtDNA) of the land snail Albinaria coerulea was determined. It contains 13 protein, two rRNA and 22 tRNA genes. Twenty-four of these genes are encoded by one and 13 genes by the other strand. The gene arrangement shares almost no similarities with that of two other molluscs for which the complete gene content and arrangement are known, the bivalve Mytilus edulis and the chiton Katharina tunicata; the protein and rRNA gene order is similar to that of another terrestrial gastropod, Cepaea nemoralis. Unusual features include the following: (1) the absence of lengthy noncoding regions (there are only 141 intergenic nucleotides interspersed at different gene borders, the longest intergenic sequence being 42 nucleotides) (2) the presence of several overlapping genes (mostly tRNAs), (3) the presence of tRNA-like structures and other stem and loop structures within genes. An RNA editing system acting on tRNAs must necessarily be invoked for posttranscriptional extension of the overlapping tRNAs. Due to these features, and also because of the small size of its genes (e.g., it contains the smallest rRNA genes among the known coelomates), it is one of the most compact mitochondrial genomes known to date.

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38

Tong, Yu, Shizhen Shen, Hui Jiang, and Zhi Chen. "Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation." Cellular Physiology and Biochemistry 43, no. 4 (2017): 1718–30. http://dx.doi.org/10.1159/000484035.

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Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations.

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39

García-López, Marta, Lydia Jiménez-Vicente, Raquel González-Jabardo, Helena Dorado, Irene Gómez-Manjón, Miguel Ángel Martín, Carmen Ayuso, Joaquín Arenas, and María Esther Gallardo. "Creation of an Isogenic Human iPSC-Based RGC Model of Dominant Optic Atrophy Harboring the Pathogenic Variant c.1861C>T (p.Gln621Ter) in the OPA1 Gene." International Journal of Molecular Sciences 25, no. 13 (June 30, 2024): 7240. http://dx.doi.org/10.3390/ijms25137240.

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Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.

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40

Lewis Luján, Lidianys María, Mark F. McCarty, James J. Di Nicolantonio, Juan Carlos Gálvez Ruiz, Ema Carina Rosas-Burgos, Maribel Plascencia-Jatomea, and Simon Bernard Iloki Assanga. "Nutraceuticals/Drugs Promoting Mitophagy and Mitochondrial Biogenesis May Combat the Mitochondrial Dysfunction Driving Progression of Dry Age-Related Macular Degeneration." Nutrients 14, no. 9 (May 9, 2022): 1985. http://dx.doi.org/10.3390/nu14091985.

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In patients with age-related macular degeneration (AMD), the crucial retinal pigment epithelial (RPE) cells are characterized by mitochondria that are structurally and functionally defective. Moreover, deficient expression of the mRNA-editing enzyme Dicer is noted specifically in these cells. This Dicer deficit up-regulates expression of Alu RNA, which in turn damages mitochondria—inducing the loss of membrane potential, boosting oxidant generation, and causing mitochondrial DNA to translocate to the cytoplasmic region. The cytoplasmic mtDNA, in conjunction with induced oxidative stress, triggers a non-canonical pathway of NLRP3 inflammasome activation, leading to the production of interleukin-18 that acts in an autocrine manner to induce apoptotic death of RPE cells, thereby driving progression of dry AMD. It is proposed that measures which jointly up-regulate mitophagy and mitochondrial biogenesis (MB), by replacing damaged mitochondria with “healthy” new ones, may lessen the adverse impact of Alu RNA on RPE cells, enabling the prevention or control of dry AMD. An analysis of the molecular biology underlying mitophagy/MB and inflammasome activation suggests that nutraceuticals or drugs that can activate Sirt1, AMPK, Nrf2, and PPARα may be useful in this regard. These include ferulic acid, melatonin urolithin A and glucosamine (Sirt1), metformin and berberine (AMPK), lipoic acid and broccoli sprout extract (Nrf2), and fibrate drugs and astaxanthin (PPARα). Hence, nutraceutical regimens providing physiologically meaningful doses of several or all of the: ferulic acid, melatonin, glucosamine, berberine, lipoic acid, and astaxanthin, may have potential for control of dry AMD.

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Hecht, Julia, Felix Grewe, and Volker Knoop. "Extreme RNA Editing in Coding Islands and Abundant Microsatellites in Repeat Sequences of Selaginella moellendorffii Mitochondria: The Root of Frequent Plant mtDNA Recombination in Early Tracheophytes." Genome Biology and Evolution 3 (January 1, 2011): 344–58. http://dx.doi.org/10.1093/gbe/evr027.

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42

Fitch, SJ, I. Bosch-Pastor, A. Antolinez, L. Gutiérrez-García, J. Marty, R. Garesse, and M. A. Fernández-Moreno. "Characterization of the mitochondrial GlutamyltRNAGln amidotransferase (GatCAB) as a new model for mitochondrial translation disorders." IBJ Plus 1, s5 (June 3, 2022): 15. http://dx.doi.org/10.24217/2531-0151.22v1s5.00015.

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Mitochondrial OXPHOS diseases are provoked by dysfunction of the OXPHOS system, showing heterogeneity from a genetic, biochemical and clinical perspective. This complexity is partly due to the involvement of proteins encoded in two genomes located in different cellular compartments, nuclear and mitochondrial DNA (mtDNA). mtDNA encodes part of the mtRNAs translation machinery, including 22 tRNAs. All these are charged by their corresponding aminoacyl-tRNA synthetases (ARS2), except mt-Gln-tRNAGln whose synthesis is carried out through an indirect pathway. The mt-tRNAGln is charged with glutamic acid (Glu) by a non-discriminating mitochondrial glutamyl-tRNA synthetase (EARS2), then the Glu is converted to Gln by a Glutamyl-tRNAGln amidotransferase, using free glutamine as an amide donor, yielding Gln- tRNAGln. This mitochondrial amidotransferase activity lies in the GatCAB complex, which is formed by three subunits: GatA (QSRL1), GatB (GATB), and GatC (GATC). Patients with mutations in these genes presented with severe cardiomyopathy and lactic acidosis, which underscores the importance of the GatCAB complex as an essential component in the translational machinery of mitochondrial protein synthesis. We have generated Knockout (KO) lines for all subunits using the genomic editing system CRISPR/Cas9 in HEK293T cells. We measured the levels of the GatCAB subunits and mitochondrial proteins by western blot. We studied the oxygen consumption levels using a Clark electrode and detected the GatCAB complex by Blue Native-western blotting. In all cases, KO cells show reduced levels of mitochondrial proteins, decreased oxygen consumption and an absence of the GatCAB complex compared to wildtype cells. In the QRSL1 and GATC KO cells the levels of all subunits diminish, but in GATB KO cells the levels of subunits A and C remain stable. Possibly, QRSL1 and GATC could form a stable but nonfunctional dimer, to which GATB would join. GATB has two potential ATG codons at the start of its sequence separated by 12 nucleotides. From over 90 clones analyzed, we only obtained one KO, by generating an upstream ORF, and two cell lines with very low expression of GATB. The latter present a duplicated region at the beginning of GATB which changes the reading frame from the first ATG; suggesting the downstream ATG as an alternative translation start codon for those ribosome small subunits that do not assemble on the first one, yielding a GATB protein missing 4 amino acids. Characterization of the molecular mechanisms that lead to the synthesis of mt-Gln-tRNAGln and its role in the physiology of the cell could allow us to comprehend the pathological manifestation of the defects in these genes and propose possible therapeutic avenues.

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Shamsnajafabadi, Hoda, Robert E. MacLaren, and Jasmina Cehajic-Kapetanovic. "Current and Future Landscape in Genetic Therapies for Leber Hereditary Optic Neuropathy." Cells 12, no. 15 (August 7, 2023): 2013. http://dx.doi.org/10.3390/cells12152013.

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Leber hereditary optic neuropathy (LHON) is the most common primary mitochondrial genetic disease that causes blindness in young adults. Over 50 inherited mitochondrial DNA (mtDNA) variations are associated with LHON; however, more than 95% of cases are caused by one of three missense variations (m.11778 G > A, m.3460 G > A, and m.14484 T > C) encoding for subunits ND4, ND1, and ND6 of the respiration complex I, respectively. These variants remain silent until further and currently poorly understood genetic and environmental factors precipitate the visual loss. The clinical course that ensues is variable, and a convincing treatment for LHON has yet to emerge. In 2015, an antioxidant idebenone (Raxone) received European marketing authorisation to treat visual impairment in patients with LHON, and since then it was introduced into clinical practice in several European countries. Alternative therapeutic strategies, including gene therapy and gene editing, antioxidant and neurotrophic agents, mitochondrial biogenesis, mitochondrial replacement, and stem cell therapies are being investigated in how effective they might be in altering the course of the disease. Allotopic gene therapies are in the most advanced stage of development (phase III clinical trials) whilst most other agents are in phase I or II trials or at pre-clinical stages. This manuscript discusses the phenotype and genotype of the LHON disease with complexities and peculiarities such as incomplete penetrance and gender bias, which have challenged the therapies in development emphasising the most recent use of gene therapy. Furthermore, we review the latest results of the three clinical trials based on adeno-associated viral (AAV) vector-mediated delivery of NADH dehydrogenase subunit 4 (ND4) with mitochondrial targeting sequence, highlighting the differences in the vector design and the rationale behind their use in the allotopic transfer.

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Bonner, Melissa, Bryan Strouse, Mindy Applegate, Paula Livingston, and Eric B. Kmiec. "DNA Damage Response Pathway and Replication Fork Stress During Oligonucleotide Directed Gene Editing." Molecular Therapy - Nucleic Acids 1 (2012): e18. http://dx.doi.org/10.1038/mtna.2012.9.

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45

Xu, Li, Piming Zhao, Andrew Mariano, and Renzhi Han. "Targeted Myostatin Gene Editing in Multiple Mammalian Species Directed by a Single Pair of TALE Nucleases." Molecular Therapy - Nucleic Acids 2 (2013): e112. http://dx.doi.org/10.1038/mtna.2013.39.

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46

Glaser, Astrid, Bradley McColl, and Jim Vadolas. "GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing." Molecular Therapy - Nucleic Acids 5 (2016): e334. http://dx.doi.org/10.1038/mtna.2016.48.

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47

Chamorro, Cristina, Angeles Mencía, David Almarza, Blanca Duarte, Hildegard Büning, Jessica Sallach, Ingrid Hausser, Marcela Del Río, Fernando Larcher, and Rodolfo Murillas. "Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes." Molecular Therapy - Nucleic Acids 5 (2016): e307. http://dx.doi.org/10.1038/mtna.2016.19.

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48

Schleifman, Erica B., Nicole Ali McNeer, Andrew Jackson, Jennifer Yamtich, Michael A. Brehm, Leonard D. Shultz, Dale L. Greiner, Priti Kumar, W. Mark Saltzman, and Peter M. Glazer. "Site-specific Genome Editing in PBMCs With PLGA Nanoparticle-delivered PNAs Confers HIV-1 Resistance in Humanized Mice." Molecular Therapy - Nucleic Acids 2 (2013): e135. http://dx.doi.org/10.1038/mtna.2013.59.

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49

Glaser, Astrid, Bradley McColl, and Jim Vadolas. "Corrigendum to GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing." Molecular Therapy - Nucleic Acids 5 (2016): e360. http://dx.doi.org/10.1038/mtna.2016.78.

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50

Palmer, Donna J., Nathan C. Grove, Jordan Ing, Ana M. Crane, Koen Venken, Brian R. Davis, and Philip Ng. "Homology Requirements for Efficient, Footprintless Gene Editing at the CFTR Locus in Human iPSCs with Helper-dependent Adenoviral Vectors." Molecular Therapy - Nucleic Acids 5 (2016): e372. http://dx.doi.org/10.1038/mtna.2016.83.

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