RESUMEN

Cytoplasmic male sterility (CMS) is of major agronomical relevance in hybrid breeding. In gametophytic CMS, abortion of pollen is determined by the grain genotype, while in sporophytic CMS, it is determined by the mother plant genotype. While several CMS mechanisms have been dissected at the molecular level, gametophytic CMS has not been straightforwardly accessible. We used the gametophytic Sha-CMS in Arabidopsis to characterize the cause and process of pollen abortion by implementing in vivo biosensing in single pollen and mitoTALEN mutagenesis. We obtained conclusive evidence that orf117Sha is the CMS-causing gene, despite distinct characteristics from other CMS genes. We measured the in vivo cytosolic ATP content in single pollen, followed pollen development, and analyzed pollen mitochondrial volume in two genotypes that differed only by the presence of the orf117Sha locus. Our results showed that the Sha-CMS is not triggered by ATP deficiency. Instead, we observed desynchronization of a pollen developmental program. Pollen death occurred independently in pollen grains at diverse stages and was preceded by mitochondrial swelling. We conclude that pollen death is grain-autonomous in Sha-CMS and propose that mitochondrial permeability transition, which was previously described as a hallmark of developmental and environmental-triggered cell death programs, precedes pollen death in Sha-CMS.

Asunto(s)

RESUMEN

BACKGROUND: The aim of this study was to evaluate and characterize the mutations induced by two TALE-based approaches, double-strand break (DSB) induction by the FokI nuclease (mitoTALEN) and targeted base editing by the DddA cytidine deaminase (mitoTALECD), to edit, for the first time, the mitochondrial genome of potato, a vegetatively propagated crop. The two methods were used to knock out the same mitochondrial target sequence (orf125). RESULTS: Targeted chondriome deletions of different sizes (236-1066 bp) were induced by mitoTALEN due to DSB repair through ectopic homologous recombination of short direct repeats (11-12 bp) present in the target region. Furthermore, in one case, the induced DSB and subsequent repair resulted in the amplification of an already present substoichiometric molecule showing a 4288 bp deletion spanning the target sequence. With the mitoTALECD approach, both nonsense and missense mutations could be induced by base substitution. The deletions and single nucleotide mutations were either homoplasmic or heteroplasmic. The former were stably inherited in vegetative offspring. CONCLUSIONS: Both editing approaches allowed us to obtain plants with precisely modified mitochondrial genomes at high frequency. The use of the same plant genotype and mtDNA region allowed us to compare the two methods for efficiency, accuracy, type of modifications induced and stability after vegetative propagation.

RESUMEN

We present two methods for enhancing the efficiency of mitochondrial DNA (mtDNA) editing in mice with DddA-derived cytosine base editors (DdCBEs). First, we fused DdCBEs to a nuclear export signal (DdCBE-NES) to avoid off-target C-to-T conversions in the nuclear genome and improve editing efficiency in mtDNA. Second, mtDNA-targeted TALENs (mitoTALENs) are co-injected into mouse embryos to cleave unedited mtDNA. We generated a mouse model with the m.G12918A mutation in the MT-ND5 gene, associated with mitochondrial genetic disorders in humans. The mutant mice show hunched appearances, damaged mitochondria in kidney and brown adipose tissues, and hippocampal atrophy, resulting in premature death.

Asunto(s)

RESUMEN

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

RESUMEN

We are living in the era of genome editing. Nowadays, targeted editing of the plant nuclear DNA is prevalent in basic biological research and crop improvement since its first establishment a decade ago. However, achieving the same accomplishment for the plant mitochondrial genome has long been deemed impossible. Recently, the pioneer studies on editing plant mitogenome have been done using the mitochondria-targeted transcription activator-like effector nucleases (mitoTALENs) in rice, rapeseed, and Arabidopsis. It is well documented that mitochondria play essential roles in plant development and stress tolerance, particularly, in cytoplasmic male sterility widely used in production of hybrids. The success of mitochondrial genome editing enables studying the fundamentals of mitochondrial genome. Furthermore, mitochondrial RNA editing (mostly by nuclear-encoded pentatricopeptide repeat (PPR) proteins) in a sequence-specific manner can simultaneously change the production of translatable mitochondrial mRNA. Moreover, direct editing of the nuclear-encoding mitochondria-targeted factors required for plant mitochondrial genome dynamics and recombination may facilitate genetic manipulation of plant mitochondria. Here, the present state of knowledge on editing the plant mitochondrial genome is reviewed.

RESUMEN

Mutations of mitochondrial DNA (mtDNA) often underlie mitochondrial disease, one of the most common inherited metabolic disorders. Since the sequencing of the human mitochondrial genome and the discovery of pathogenic mutations in mtDNA more than 30 years ago, a movement towards generating methods for robust manipulation of mtDNA has ensued, although with relatively few advances and some controversy. While developments in the transformation of mammalian mtDNA have stood still for some time, recent demonstrations of programmable nuclease-based technology suggest that clinical manipulation of mtDNA heteroplasmy may be on the horizon for these largely untreatable disorders. Here we review historical and recent developments in mitochondrially targeted nuclease technology and the clinical outlook for treatment of hereditary mitochondrial disease.

Asunto(s)

RESUMEN

Most patients with mitochondrial DNA (mtDNA) mutations have a mixture of mutant and wild-type mtDNA in their cells. This phenomenon, known as mtDNA heteroplasmy, provides an opportunity to develop therapies by selectively eliminating the mutant fraction. In the last decade, several enzyme-based gene editing platforms were developed to cleave specific DNA sequences. We have taken advantage of these enzymes to develop reagents to selectively eliminate mutant mtDNA. The replication of intact mitochondrial genomes normalizes mtDNA levels and consequently mitochondrial function. In this chapter, we describe the methodology used to design and express these nucleases in mammalian cells in culture and in vivo.

Asunto(s)

RESUMEN

In recent years mitochondrial DNA (mtDNA) has transitioned to greater prominence across diverse areas of biology and medicine. The recognition of mitochondria as a major biochemical hub, contributions of mitochondrial dysfunction to various diseases, and several high-profile attempts to prevent hereditary mtDNA disease through mitochondrial replacement therapy have roused interest in the organellar genome. Subsequently, attempts to manipulate mtDNA have been galvanized, although with few robust advances and much controversy. Re-engineered protein-only nucleases such as mtZFN and mitoTALEN function effectively in mammalian mitochondria, although efficient delivery of nucleic acids into the organelle remains elusive. Such an achievement, in concert with a mitochondria-adapted CRISPR/Cas9 platform, could prompt a revolution in mitochondrial genome engineering and biological understanding. However, the existence of an endogenous mechanism for nucleic acid import into mammalian mitochondria, a prerequisite for mitochondrial CRISPR/Cas9 gene editing, remains controversial.

Asunto(s)

RESUMEN

Mutations in mtDNA lead to muscular and neurological diseases and are linked to aging. The most frequent aberrancy is the "common deletion" that involves a 4,977-bp region flanked by 13-bp repeats. To investigate the basis of this deletion, we developed a single-molecule mtDNA combing method. The analysis of replicating mtDNA molecules provided in vivo evidence in support of the asymmetric mode of replication. Furthermore, we observed frequent fork stalling at the junction of the common deletion, suggesting that impaired replication triggers the formation of this toxic lesion. In parallel experiments, we employed mito-TALENs to induce breaks in distinct loci of the mitochondrial genome and found that breaks adjacent to the 5' repeat trigger the common deletion. Interestingly, this process was mediated by the mitochondrial replisome independent of canonical DSB repair. Altogether, our data underscore a unique replication-dependent repair pathway that leads to the mitochondrial common deletion.

Asunto(s)

RESUMEN

For more than a decade, mitochondria-targeted nucleases have been used to promote double-strand breaks in the mitochondrial genome. This was done in mitochondrial DNA (mtDNA) homoplasmic systems, where all mtDNA molecules can be affected, to create models of mitochondrial deficiencies. Alternatively, they were also used in a heteroplasmic model, where only a subset of the mtDNA molecules were substrates for cleavage. The latter approach showed that mitochondrial-targeted nucleases can reduce mtDNA haplotype loads in affected tissues, with clear implications for the treatment of patients with mitochondrial diseases. In the last few years, designer nucleases, such as ZFN and TALEN, have been adapted to cleave mtDNA, greatly expanding the potential therapeutic use. This chapter describes the techniques and approaches used to test these designer enzymes.

Asunto(s)