Mitochondrial medicine in the omics era

Rahman, Joyeeta; Rahman, Shamima

doi:10.1016/s0140-6736(18)30727-x

Cited by 224 publications

(198 citation statements)

References 145 publications

(162 reference statements)

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“…This indicates that the lack of effect on mtDNA levels using mutant forms of Su9LbCas12a-GFP was not due to a lower crRNA transfection or expression efficiency. Moreover, MT-TL1 and COXII mRNA levels also increased after co-transfection with WT Su9LbCas12a-GFP and crRNA (2) (Figures S8F,G). In a further validation experiment to confirm the increase in COXII DNA levels after co-transfection with WT Su9LbCas12a-GFP and gRNA (2), no significant changes in mtDNA levels were observed (Figure S8H).…”

Section: Analysis Of Mitochondrial Lbcas12a Cleavage Activity and Mtdmentioning

confidence: 91%

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Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

Antón

Mullally

Ford

et al. 2020

Preprint

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Current methodologies for targeting the mitochondrial genome for basic research and/or therapeutic strategy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. The development of a functional molecular toolbox for CRISPR-mediated mitochondrial genome editing is therefore desirable, as this could enable precise targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes; however, published reports of "MitoCRISPR" systems have, to date, lacked reproducibility and independent corroboration. Here, we have explored the requirements for a functional MitoCRISPR system in human cells by engineering several versions of CRISPR nucleases, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of gRNA modifications that reportedly induce mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and influences 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. Finally, we present evidence linking 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 if MitoCRISPR is to be realised.100% of mtDNA copies carry the mutation, or heteroplasmic, where the mutation is carried by a subset of the total mtDNA. In general, mutation load above ~70% is required to present a severe phenotype, although this threshold is disease specific. As a treatment to selectively degrade disease-causing mtDNA haplotypes, the field has developed targeted endonucleases which produce dsDNA breaks in the mutated mtDNA copies, which are then degraded by the mitochondria rather than being repaired [5,6]. The remaining wild type mtDNA then replicates to re-establish mtDNA copy number. This "heteroplasmy purification" has been successfully demonstrated in cell culture and in mouse models using restriction endonucleases (MitoREs), zinc finger nucleases (MitoZFNs), TALENs (MitoTALENs), and homing endonucleases [7,8]. However, although independently validated [9], these tools are impracticable and not widely adopted, because: (i) REs match few clinical mutations and cannot be readily re-engineered [10], and they additionally have high "off-target" cleavage rates [11,12]; (ii) ZFNs require rounds of protein engineering/refinement; (iii) assembly of TALE parts is hindered by problematic cloning of DNA repeats;(iv) TALENs and ZFNs can have low import efficiency and can be mis-trafficked (e.g. ZFs have internal nuclear targeting sequences) [13][14][15]. To overcome some of these difficulties, we have investigated the use of a re-engineered flexible version of the CRISPR method for mitochondrial gen...

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Section: Analysis Of Mitochondrial Lbcas12a Cleavage Activity and Mtdmentioning

confidence: 91%

“…The development of a reliable MitoCRISPR system requires ( Figure 1A): (1) the targeted delivery of a CRISPR nuclease to the mitochondria by fusion to a peptide mitochondrial targeting sequence (MTS); (2) mitochondrial targeting of a CRISPR gRNA, possibly through fusion to a mitochondrial targeting domain;…”

Section: Design Of a Mitocrispr Systemmentioning

confidence: 99%

Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

Antón

Mullally

Ford

et al. 2020

Preprint

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“…The term primary mitochondrial disease is generally accepted to refer to a large group of heterogeneous inborn errors of metabolism that directly or indirectly affect the integrity and/or function of mitochondria, particularly the OXPHOS system. 68 CFD was first reported in the context of mitochondrial disease in 1983 in a young woman with Kearns-Sayre syndrome (KSS, OMIM #530000) associated with ragged-red fibre myopathy, progressive neurological decline and white matter lesions and basal ganglia calcification on computed tomography of the brain. 69 In the intervening 35 years CFD has been recognized as a relatively frequent occurrence in primary mitochondrial disease, although the exact prevalence remains unknown.…”

Section: Mitochondrial Disordersmentioning

confidence: 99%

Cerebral folate deficiency: Analytical tests and differential diagnosis

Pope

Artuch

Heales

et al. 2019

J of Inher Metab Disea

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Cerebral folate deficiency is typically defined as a deficiency of the major folate species 5‐methyltetrahydrofolate in the cerebrospinal fluid (CSF) in the presence of normal peripheral total folate levels. However, it should be noted that cerebral folate deficiency is also often used to describe conditions where CSF 5‐MTHF is low, in the presence of low or undefined peripheral folate levels. Known defects of folate transport are deficiency of the proton coupled folate transporter, associated with systemic as well as cerebral folate deficiency, and deficiency of the folate receptor alpha, leading to an isolated cerebral folate deficiency associated with intractable seizures, developmental delay and/or regression, progressive ataxia and choreoathetoid movement disorders. Inborn errors of folate metabolism include deficiencies of the enzymes methylenetetrahydrofolate reductase, dihydrofolate reductase and 5,10‐methenyltetrahydrofolate synthetase. Cerebral folate deficiency is potentially a treatable condition and so prompt recognition of these inborn errors and initiation of appropriate therapy is of paramount importance. Secondary cerebral folate deficiency may be observed in other inherited metabolic diseases, including disorders of the mitochondrial oxidative phosphorylation system, serine deficiency, and pyridoxine dependent epilepsy. Other secondary causes of cerebral folate deficiency include the effects of drugs, immune response activation, toxic insults and oxidative stress. This review describes the absorption, transport and metabolism of folate within the body; analytical methods to measure folate species in blood, plasma and CSF; inherited and acquired causes of cerebral folate deficiency; and possible treatment options in those patients found to have cerebral folate deficiency.

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“…All remaining protein components of the mitochondrial gene maintenance and expression machineries such as proteins responsible for mtDNA transcription, precursor RNA processing enzymes, the mitoribosomal proteins, mitochondrial aminoacyl tRNA synthetases, and others are encoded by the nuclear genes (Hallberg & Larsson, ; Rorbach & Minczuk, ). More than 50 nuclear‐encoded mitochondrial proteins involved in mitochondrial gene expression are linked to heritable disorders (Frazier, Thorburn, & Compton, ; Powell, Nicholls, & Minczuk, ; Rahman & Rahman, ; Stenton & Prokisch, ; Van Haute et al, ).…”

Section: Introductionmentioning

confidence: 99%

Mutations inELAC2associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3′‐end processing

et al. 2019

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Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3′ ends of mitochondrial pre‐tRNAs. Here, we report the identification of 16 novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy (HCM), and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modeled the residues affected by a missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile‐onset forms of HCM and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism

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Mitochondrial medicine in the omics era

Cited by 224 publications

References 145 publications

Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

Cerebral folate deficiency: Analytical tests and differential diagnosis

Mutations inELAC2associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3′‐end processing

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