RESUMO
The development of curative treatments for mitochondrial diseases, which are often caused by mutations in mitochondrial DNA (mtDNA) that impair energy metabolism and other aspects of cellular homoeostasis, is hindered by an incomplete understanding of the underlying biology and a scarcity of cellular and animal models. Here we report the design and application of a library of double-stranded-DNA deaminase-derived cytosine base editors optimized for the precise ablation of every mtDNA protein-coding gene in the mouse mitochondrial genome. We used the library, which we named MitoKO, to produce near-homoplasmic knockout cells in vitro and to generate a mouse knockout with high heteroplasmy levels and no off-target edits. MitoKO should facilitate systematic and comprehensive investigations of mtDNA-related pathways and their impact on organismal homoeostasis, and aid the generation of clinically meaningful in vivo models of mtDNA dysfunction.
Assuntos
Edição de Genes , Genoma Mitocondrial , Camundongos , Animais , Genoma Mitocondrial/genética , DNA Mitocondrial/genética , Mutação , Biblioteca GênicaRESUMO
The human mitochondrial genome must be replicated and expressed in a timely manner to maintain energy metabolism and supply cells with adequate levels of adenosine triphosphate. Central to this process is the idea that replication primers and gene products both arise via transcription from a single light strand promoter (LSP) such that primer formation can influence gene expression, with no consensus as to how this is regulated. Here, we report the discovery of a second light strand promoter (LSP2) in humans, with features characteristic of a bona fide mitochondrial promoter. We propose that the position of LSP2 on the mitochondrial genome allows replication and gene expression to be orchestrated from two distinct sites, which expands our long-held understanding of mitochondrial gene expression in humans.
Assuntos
Genoma Mitocondrial , Trifosfato de Adenosina/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transcrição GênicaRESUMO
Mitochondria host key metabolic processes vital for cellular energy provision and are central to cell fate decisions. They are subjected to unique genetic control by both nuclear DNA and their own multi-copy genome - mitochondrial DNA (mtDNA). Mutations in mtDNA often lead to clinically heterogeneous, maternally inherited diseases that display different organ-specific presentation at any stage of life. For a long time, genetic manipulation of mammalian mtDNA has posed a major challenge, impeding our ability to understand the basic mitochondrial biology and mechanisms underpinning mitochondrial disease. However, an important new tool for mtDNA mutagenesis has emerged recently, namely double-stranded DNA deaminase (DddA)-derived cytosine base editor (DdCBE). Here, we test this emerging tool for in vivo use, by delivering DdCBEs into mouse heart using adeno-associated virus (AAV) vectors and show that it can install desired mtDNA edits in adult and neonatal mice. This work provides proof-of-concept for use of DdCBEs to mutagenize mtDNA in vivo in post-mitotic tissues and provides crucial insights into potential translation to human somatic gene correction therapies to treat primary mitochondrial disease phenotypes.
Assuntos
DNA Mitocondrial/genética , Edição de Genes/métodos , Genes Mitocondriais/genética , Terapia Genética/métodos , Doenças Mitocondriais/terapia , Animais , Dependovirus/genética , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Mitocôndrias/genética , Doenças Mitocondriais/genética , Modelos Animais , Mutagênese , Mutação , Estudo de Prova de ConceitoRESUMO
Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterized a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, associated with less accurate mitochondrial tRNASer(AGY) processing from the primary transcript and a defect in the maturation of the mitoribosomal small subunit. Our results suggest that YbeY performs a dual, likely independent, function in mitochondria being involved in precursor RNA processing and mitoribosome biogenesis. Issue Section: Nucleic Acid Enzymes.
