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

2020 ◽  
Author(s):  
Zuriñe Antón ◽  
Grace Mullally ◽  
Holly Ford ◽  
Marc W. van der Kamp ◽  
Mark D. Szczelkun ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Copy Number ◽  
Protein Targeting ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Mitochondrial Diseases ◽  
Basic Research ◽  
Strategy Development ◽  
Mtdna Copy Number ◽  
Mitochondrial Import

ABSTRACTCurrent 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.

scholarly journals Mitochondrial import, health and mtDNA copy number variability seen when using type II and type V CRISPR effectors

2020 ◽  
Vol 133 (18) ◽  
pp. jcs248468 ◽  
Author(s):  
Zuriñe Antón ◽  
Grace Mullally ◽  
Holly C. Ford ◽  
Marc W. van der Kamp ◽  
Mark D. Szczelkun ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Copy Number ◽  
Protein Targeting ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Mitochondrial Diseases ◽  
Mtdna Copy Number ◽  
Data Link ◽  
Mitochondrial Import ◽  
Type V

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.

scholarly journals Study of the effect of the introduction of mitochondrial import determinants into the gRNA structure on the activity of the gRNA/SpCas9 complex in vitro

2020 ◽  
Vol 24 (5) ◽  
pp. 512-518
Author(s):  
E. G. Zakirova ◽  
Y. V. Vyatkin ◽  
N. A. Verechshagina ◽  
V. V. Muzyka ◽  
I. O. Mazunin ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Copy Number ◽  
Clinical Symptoms ◽  
Mitochondrial Diseases ◽  
Wild Type ◽  
Therapeutic Approaches ◽  
Mitochondrial Import ◽  
Promising Tool ◽  
First Time

It has long been known that defects in the structure of the mitochondrial genome can cause various neuromuscular and neurodegenerative diseases. Nevertheless, at present there is no effective method for treating mitochondrial diseases. The major problem with the treatment of such diseases is associated with mitochondrial DNA (mtDNA) heteroplasmy. It means that due to a high copy number of the mitochondrial genome, mutant copies of mtDNA coexist with wild-type molecules in the same organelle. The clinical symptoms of mitochondrial diseases and the degree of their manifestation directly depend on the number of mutant mtDNA molecules in the cell. The possible way to reduce adverse effects of the mutation is by shifting the level of heteroplasmy towards the wild-type mtDNA molecules. Using this idea, several gene therapeutic approaches based on TALE and ZF nucleases have been developed for this purpose. However, the construction of protein domains of such systems is rather long and laborious process. Meanwhile, the CRISPR/Cas9 system is fundamentally different from protein systems in that it is easy to use, highly efficiency and has a different mechanism of action. All the characteristics and capabilities of the CRISPR/Cas9 system make it a promising tool in mitochondrial genetic engineering. In this article, we demonstrate for the first time that the modification of gRNA by integration of specific mitochondrial import determinants in the gRNA scaffold does not affect the activity of the gRNA/Cas9 complex in vitro.

86 Effect of invitro culture conditions on mitochondria functions in mouse embryos

2020 ◽  
Vol 32 (2) ◽  
pp. 169
Author(s):  
M. Czernik ◽  
D. Winiarczyk ◽  
S. Sampino ◽  
P. Greda ◽  
J. A. Modlinski ◽  
...  
Keyword(s):  
Embryo Development ◽  
Copy Number ◽  
Energy Demand ◽  
Mitochondrial Membrane ◽  
Mitochondrial Dynamics ◽  
Culture Conditions ◽  
Mouse Embryos ◽  
Mtdna Copy Number ◽  
Mitochondrial Functions ◽  
Mitochondria Functions

Mitochondria provide the energy for oocyte maturation, fertilisation, and embryo formation via oxidative phosphorylation. Consequently, any adverse influence on mitochondrial function may negatively affect the development of pre-implantation embryos especially because there is no mitochondrial DNA (mtDNA) replication until post-implantation. Studies in the field of mitochondrial dynamics have identified an intriguing link between energy demand/supply balance and mitochondrial architecture, which may suggest that inappropriate culture conditions may inhibit mitochondrial functions, which may negatively affect embryo development. We wanted to check whether invitro culture (IVC) conditions of mouse embryos affect mitochondrial functionality. The IVC as well as naturally matted (NM) mouse embryos at the 2-cell and blastocyst stage were subjected to mitochondrial analysis (distribution, organisation, and mitochondrial membrane potential), and expression of mRNA and proteins involved in regulation of mitochondria functions, as well as number of mtDNA copies, were evaluated. Significance level was set at 0.05. We observed that the mitochondria in 2-cell IVC embryos were less numerous and localised mainly in the pericortical region of the cytoplasm, whereas mitochondria in NM embryos were numerous and homogeneously distributed in both blastomeres. Drastic differences were observed in blastocysts. Mitochondria in the IVC group were fragmented, rounded, and aggregated mainly in the perinuclear region of the cells, whereas mitochondria of NM blastocysts were numerous and created an elongated mitochondrial network along the cells. Time-lapse analysis showed reduced mitochondrial and mitochondrial membrane activity in IVC blastocysts. Moreover, our results indicate the IVC group had reduced mRNA expression of mitofusin 1, mitofusin 2, and optic atrophy 1 responsible for mitochondrial fusion. Additionally, mtDNA copy number for IVC blastocysts (398 887.45±30 608.65) was significantly lower than that of NM blastocysts (593 367.12±66 540.32; P<0.02). Furthermore, no significant differences were found in mtDNA copy number of IVC 2-cell embryos when compared with NM embryos. The results obtained clearly showed that IVC conditions affect proper mitochondrial functionality and hence embryo development.

Mitochondrial Genome Instability in AML Is Associated with Reduction in the Mitochondrial Replication.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2754-2754
Author(s):  
Myung-Geun Shin ◽  
Hyeoung-Joon Kim ◽  
Hye-Ran Kim ◽  
Il-Kwon Lee ◽  
Duck Cho ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Control Region ◽  
Buccal Mucosa ◽  
Copy Number ◽  
Genome Instability ◽  
Point Mutations ◽  
Mtdna Copy Number ◽  
Control Subjects ◽  
Length Changes ◽  
Mtdna Variants

Abstract Genetic changes in mitochondrial DNA (mtDNA) have been hypothesized more widely to play important roles in senescence, autoimmune disease and malignancy because of a paucity of introns and limited repair mechanisms. Malfunction of mismatch repair genes produces genome instability which plays an important role in the development of human cancers. The mtDNA markers for mitochondrial genome instability (mtGI) were point mutations, insertions, deletions and length changes in homopolymeric nucleotide tracts. We investigated the mtGI in AML cells and its effect to alteration in mtDNA copy number. Forty-eight matched AML bone marrow and buccal mucosa samples, and blood samples from 57 control subjects were collected after receiving Institutional Review Board approval and informed consent. We directly sequenced the control region, the tRNA leucine 1 gene plus a part of NADH dehydrogenase (ND)1 and cytochrome b (CYTB) of mtDNA. In an attempt to investigate mtGI, we carried out a qualitative and quantitative profiling mtDNA length heteroplasmies of six mtGIs (np 303–315 poly C, np 16184–16193 poly C, np 514–511 CA repeats, np 3566–3572 poly C, np 12385–12391 poly C and np 12418–12426 poly A) using a size-based PCR product separation by capillary electrophoresis (ABI Prism Genotyper version 3.1). Length heteroplasmy was further confirmed by cloning and sequencing. Quantitative analysis of mtDNA molecules was performed using the QuantiTect SYBR Green PCR kit (Qiagen). In the current study, we detected a large number of polymorphisms as well as new mtDNA variants. A total of 606 mtDNA sequence variants were identified. Among these, 15 mtDNA variants were identified as novel mutations that were absent from corresponding buccal mucosa, control subjects and established mtDNA polymorphism databases. In the control region, we found two types of mtDNA alterations - base substitutions and small deletions/insertions as well as the length heteroplasmies in the np 303 to 315 poly-C, np 16184 to 16193 poly-C and 514–515 CA repeats. Seven patients (15%) had leukemia cell-specific mtDNA substitution mutations in the ND1 and CYTB genes. Somatic mtDNA control region mutations found in this study preferentially altered known mtDNA regulatory elements. AML cells had about a two-fold decrease in mtDNA copy number compared with the results from control subjects (63 x 106 molecules/ul ± 23 x 106 vs 122 x 106 molecules/ul ± 73 x 106). Our results are consistent with a recent observation that carcinogenesis in the liver, kidney and lung involves a decrease of the cellular mitochondrial content and decreased mtDNA copy number (Mutat Res2004;547:71–78). In conclusion, mtGI including point mutations, length changes (insertions or deletions) in homopolymeric tracts commonly occurred in AML cells and reduction in mtDNA copy number may result from either mtDNA control region mutations or impairment of mitochondrial biogenesis. These findings suggest that biogenesis of mitochondria is repressed in the leukemogenesis process of the human hematopoietic tissue.

Role of the mitochondrial genome in preimplantation development and assisted reproductive technologies

2005 ◽  
Vol 17 (2) ◽  
pp. 15 ◽  
Author(s):  
Lawrence C. Smith ◽  
Jacob Thundathil ◽  
France Filion
Keyword(s):  
Mitochondrial Genome ◽  
Copy Number ◽  
Cell Function ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Domestic Animals ◽  
Preimplantation Development ◽  
Mtdna Copy Number ◽  
Transcription And Replication

Our fascination for mitochondria relates to their origin as symbiotic, semi-independent organisms on which we, as eukaryotic beings, rely nearly exclusively to produce energy for every cell function. Therefore, it is not surprising that these organelles play an essential role in many events during early development and in artificial reproductive technologies (ARTs) applied to humans and domestic animals. However, much needs to be learned about the interactions between the nucleus and the mitochondrial genome (mtDNA), particularly with respect to the control of transcription, replication and segregation during preimplantation. Nuclear-encoded factors that control transcription and replication are expressed during preimplantation development in mice and are followed by mtDNA transcription, but these result in no change in mtDNA copy number. However, in cattle, mtDNA copy number increases during blastocyst expansion and hatching. Nuclear genes influence the mtDNA segregation patterns in heteroplasmic animals. Because many ARTs markedly modify the mtDNA content in embryos, it is essential that their application is preceded by careful experimental scrutiny, using suitable animal models.

scholarly journals Presequence-Independent Mitochondrial Import of DNA Ligase Facilitates Establishment of Cell Lines with Reduced mtDNA Copy Number

PLoS ONE ◽  
2016 ◽  
Vol 11 (3) ◽  
pp. e0152705 ◽  
Author(s):  
Domenico Spadafora ◽  
Natalia Kozhukhar ◽  
Mikhail F. Alexeyev
Keyword(s):  
Cell Lines ◽  
Copy Number ◽  
Dna Ligase ◽  
Mtdna Copy Number ◽  
Mitochondrial Import

scholarly journals Role of the Mitochondrial Protein Import Machinery and Protein Processing in Heart Disease

2021 ◽  
Vol 8 ◽  
Author(s):  
Fujie Zhao ◽  
Ming-Hui Zou
Keyword(s):  
Heart Disease ◽  
Protein Import ◽  
Protein Quality ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Nuclear Genome ◽  
Major Protein ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Mitochondrial Import

Mitochondria are essential organelles for cellular energy production, metabolic homeostasis, calcium homeostasis, cell proliferation, and apoptosis. About 99% of mammalian mitochondrial proteins are encoded by the nuclear genome, synthesized as precursors in the cytosol, and imported into mitochondria by mitochondrial protein import machinery. Mitochondrial protein import systems function not only as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles. Mitochondrial protein import deficiency is linked to various diseases, including cardiovascular disease. In this review, we describe an emerging class of protein or genetic variations of components of the mitochondrial import machinery involved in heart disease. The major protein import pathways, including the presequence pathway (TIM23 pathway), the carrier pathway (TIM22 pathway), and the mitochondrial intermembrane space import and assembly machinery, related translocases, proteinases, and chaperones, are discussed here. This review highlights the importance of mitochondrial import machinery in heart disease, which deserves considerable attention, and further studies are urgently needed. Ultimately, this knowledge may be critical for the development of therapeutic strategies in heart disease.

Mitochondrial Genome Changes As a Measure Of Iron-Induced Mitochondrial Stress In Transfusion-Dependent Thalassemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2256-2256
Author(s):  
Esteban Gomez ◽  
Cassandra Calloway ◽  
Sang Hoon Lee ◽  
Jay Kim ◽  
Navpreet Dhillon ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Iron Overload ◽  
Copy Number ◽  
Complex I ◽  
Dry Weight ◽  
Myocardial Iron ◽  
Mtdna Copy Number ◽  
Complex Iv ◽  
Mitochondrial Stress ◽  
Complex I Activity

Abstract Introduction Iron overload is a consequence of chronic blood transfusions to treat severe forms of thalassemia. While patient outcomes have improved with advances in iron overload assessment and chelation therapy, iron toxicity continues to be the primary determinant of morbidity. Mitochondrial dysfunction is of central importance in iron overload. However, clinical assessment of mitochondrial damage has not been feasible because current methods require invasive tissue sampling. Methods Novel bioassays were used to assess changes in the mitochondrial genome from a small amount of peripheral blood. We studied the relationship between mitochondrial DNA (mtDNA), mitochondrial respiratory complexes, and clinical measures of iron overload. Real-time PCR-based assays were used to measure mtDNA copy number per nuclear DNA copy (mtDNA:nDNA) and the frequency of the mtDNA 4977-bp deletion (ΔmtDNA4977). The quantity of mitochondrial respiratory complex I and IV in blood was measured using ELISA and expressed as a percentage of quantity in HepG2 cells. Results 44 individuals with thalassemia (median age 34.9 years, range 14 to 52.4 years) and 27 healthy controls (median age 22 years, range 19 to 39 years) were studied in this analysis. The median liver iron concentration (LIC) in thalassemia was 11.0 mg/g dry-weight (2.3-59.4 mg/g dry-weight), while the median ferritin was 2220 mcg/L (308-9470 mcg/L). The median cardiac T2* was 28.7 ms (6.5-45.8 ms). The mtDNA:nDNA was estimated and the median mtDNA copy number was 249.8 (87.6-738.3) in thalassemia, compared with 225.4 (101.9-348.7) in controls. The mtDNA copy number was significantly elevated in thalassemia (p=0.033, Mann-Whitney test). The mtDNA copy number increased with age in thalassemia (r=0.45, p=0.005), but no correlation with age was observed in the control group (r=0.06, p=n.s.). Myocardial iron deposition influenced the mtDNA copy number as evidenced by an inverse relationship with T2* (r= -0.45, p=0.006). The frequency of the ΔmtDNA4977deletion was analyzed in a subset of subjects and was found to be 8-fold higher in thalassemia versus controls (p=0.012). The median quantity of complex IV in thalassemia was 3.4% (0 to 104.2) compared with 2.5% (range 0 to 18.67) in controls. The median complex I activity in thalassemia was 6.3% (range 0 to 161.4) compared with 17.86% (range 0 to 55.0) in controls. These differences were not statistically significantly between the two groups. Higher values of mtDNA copy number were associated with increased quantity of complex IV (r=0.515, p=0.10) in thalassemia, but not in controls. No correlation of mtDNA copy number was observed with complex I activity. LIC, ferritin, and platelet count did not influence the mtDNA:nDNA ratios. Interpretation Alterations in mtDNA can be measured in blood samples of patients with thalassemia and iron overload. Increases in mitochondrial genome copy number and ΔmtDNA4977 deletion frequency may be a reflection of iron-induced mitochondrial stress. This hypothesis is supported by an increase in mtDNA copy number with age in thalassemia, but not in controls. The relation between mtDNA changes and myocardial iron suggests that this assay may possess physiological relevance. These preliminary results support the potential application of these non-invasive assays for the assessment of iron-induced organ damage. Disclosures: No relevant conflicts of interest to declare.

Stable heteroplasmy but differential inheritance of a large mitochondrial DNA deletion in nematodes

2002 ◽  
Vol 80 (5) ◽  
pp. 645-654 ◽  
Author(s):  
William Y Tsang ◽  
Bernard D Lemire
Keyword(s):  
Mitochondrial Dna ◽  
Caenorhabditis Elegans ◽  
Copy Number ◽  
Mitochondrial Diseases ◽  
Wild Type ◽  
Mtdna Copy Number ◽  
Mitochondrial Dna Deletion ◽  
Dna Deletion ◽  
Average Proportion ◽  
Mtdna Deletion

Many human mitochondrial diseases are associated with defects in the mitochondrial DNA (mtDNA). Mutated and wild-type forms of mtDNA often coexist in the same cell in a state called heteroplasmy. Here, we report the isolation of a Caenorhabditis elegans strain bearing the 3.1-kb uaDf5 deletion that removes 11 genes from the mtDNA. The uaDf5 deletion is maternally transmitted and has been maintained for at least 100 generations in a stable heteroplasmic state in which it accounts for ~60% of the mtDNA content of each developmental stage. Heteroplasmy levels vary between individual animals (from ~20 to 80%), but no observable phenotype is detected. The total mtDNA copy number in the uaDf5 mutant is approximately twice that of the wild type. The maternal transmission of the uaDf5 mtDNA is controlled by at least two competing processes: one process promotes the increase in the average proportion of uaDf5 mtDNA in the offspring, while the second promotes a decrease. These two forces prevent the segregation of the mtDNAs to homoplasmy.Key words: mtDNA deletion, Caenorhabditis elegans, heteroplasmy, inheritance, mtDNA copy number.

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