Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria [Exp2_POLGD274A_MGME1KO_ScaI]
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1158714 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30608515_1.fastq.gz | Fastqsanger.gz | |||
| SRR30608515_2.fastq.gz | Fastqsanger.gz | |||
| SRR30608516_1.fastq.gz | Fastqsanger.gz | |||
| SRR30608516_2.fastq.gz | Fastqsanger.gz | |||
| SRR30608517_1.fastq.gz | Fastqsanger.gz |
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Similar Datasets
Project description:Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria [Exp3_MGME1KO_KL_iApaLI]
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Project description:Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria [DOGMAseq]
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Project description:Engineering mtDNA Deletions by Reconstituting End-Joining in Human Mitochondria [Exp1_iScaI_KL_T4]
| PRJNA1158713 | ENA
Project description:Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial disorders and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a ~3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of ~75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies.
2025-03-10 | GSE276696 | GEO
Project description:Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial disorders and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a ~3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of ~75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies.
2025-03-10 | GSE276694 | GEO
Project description:Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial disorders and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a ~3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of ~75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies.
2025-03-10 | GSE276718 | GEO
Project description:Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled the precise introduction of base substitutions and the effective removal of genomes carrying harmful mutations. However, the reconstitution of mtDNA deletions responsible for severe mitochondrial disorders and age-related diseases has not yet been achieved in human cells. Here, we developed a method to engineer specific mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. As a proof-of-concept, we used mito-EJ and mito-ScaI to generate a panel of clonal cell lines harboring a ~3.5 kb mtDNA deletion with the full spectrum of heteroplasmy. Investigating these isogenic cells revealed a critical threshold of ~75% deleted genomes, beyond which cells exhibited depletion of OXPHOS proteins, severe metabolic disruption, and impaired growth in galactose-containing media. Single-cell multiomic analysis revealed two distinct patterns of nuclear gene deregulation in response to mtDNA deletion accumulation; one triggered at the deletion threshold and another progressively responding to increasing heteroplasmy. In summary, the co-expression of mito-EJ and programable nucleases provides a powerful tool to model disease-associated mtDNA deletions in different cell types. Establishing a panel of cell lines with a large-scale deletion at varying levels of heteroplasmy is a valuable resource for understanding the impact of mtDNA deletions on diseases and guiding the development of potential therapeutic strategies.
2025-03-10 | GSE276699 | GEO
Project description:Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA.
2018-07-11 | PXD009138 | Pride
Project description:Aim: To determine whether mtDNA deletions arise upon the loss of mitochondria fusion in heart tissue Method: DNA was isolated heart mitochondria from control and heart-specific double mitofusin1-2 (dMfn) KO animals and used to generate libraries for sequencing to detect mtDNA deletions. Total genomic DNA from the Deletor mouse was provided by Anu Suomalainen-Wartiovaara and used as a positive control for the detection of mtDNA rearrangements. A standard Illumina TrueSeq paired-end library was prepared with ~500 base pair fragment inserts. Paired-end 100 base pair sequencing was conducted using an Illumina HiSeq 2500. The reads were mapped to the genomic sequence without the mitochondria using bowtie (VN: 2.1.0) to remove nuclear-genomic sequences. The unmapped reads were then mapped to the mitochondrial sequence (GenBank JF286601.1) using bwa (n=0.04, VN: 0.6.2-r126) with unmapped reads undergoing an additional mapping round after trimming fastx_trimmer, VN: 0.0.13.2) to ensure higher mapping results. Using samtools (VN: 1.0: samtools –f1 –F14) reads, where the two paired sequences were observed to be greater than 600 base pair apart were identified as those containing deletion breakpoints. These reads were extracted for additional analysis. Results: Control and dMfn KO samples showed similar frequencys of breakpoints, while the positive control (Deletor mouse) showed a high frequecny of large breakpoints.
2018-12-28 | GSE124420 | GEO
Project description:Purpose: To develop a pipeline (Splice-Break) for high-resolution quantification of mtDNA deletions, provide a catalogue of human mtDNA deletion breakpoints, and evaluate mtDNA deletions in brains from subjects with psychiatric disorders. Methods: 93 samples from human postmortem brain and blood were obtained from the Southwest Brain Bank (SBB) and University of California, Irvine (UCI) Brain Bank. Total DNA was extracted from frozen homogenate tissue and mtDNA was amplified/enriched using a single long-range PCR. Mitochondrial amplicons were purified by bead purification to retain both wild-type and deleted molecules (i.e., no gel excision was performed). mtDNA-enriched PCR amplicons were prepared for sequencing using standard Illumina protocols for DNA. Samples were sequenced 150-mer paired-end reads, in multiplex (96x per lane), on an unpatterned flowcell (HiSeq 2500). Fastq files were processed using our Splice-Break pipeline for the detection and relative quantification of mtDNA deletions. Results: A catalogue of 4,489 putative mitochondrial DNA (mtDNA) deletions, including their frequency and relative read rate, was produced. Analyses of 93 samples from postmortem brain and blood found 1) the 4,977bp “common deletion” was neither the most frequent deletion nor the most abundant; 2) brain contained significantly more mtDNA deletions than blood; 3) many high frequency deletions were previously reported in MitoBreak, suggesting they are present at low levels in metabolically active tissues and are not exclusive to individuals with diagnosed mitochondrial pathologies; 4) many individual deletions (and cumulative deletion metrics) had significant and positive correlations with age; and 5) the highest deletion burdens were observed in a subset of subjects with major depressive disorder (MDD), and these subjects had mtDNA deletion levels at or above those detected in typical deletion pathologies (e.g., Kearns-Sayre syndrome (KSS) muscle). Conclusions: Collectively, these data suggest the Splice-Break pipeline can detect and quantify mtDNA deletions at a high level of resolution.
2018-11-01 | GSE118615 | GEO