Biology subjects

Mazunin, I.

Publications and source records attributed to Mazunin, I..

9 recordsLinked to original sources

bioRxiv2025

Targeting of human mitochondrial DNA with programmable pAgo nuclease

Manipulating the mitochondrial genome remains a major challenge in genetic engineering, largely due to the mitochondrial double-membrane structure. While recent advances have expanded the genetic toolkit for nuclear and cytoplasmic targets, precise editing of mtDNA has remained elusive. Here we report the first successful mitochondrial import of a functional RNA-guided prokaryotic Argonaute protein from the mesophilic bacterium Alteromonas macleodii (AmAgo). By directing AmAgo to the single-stranded D- or R-loop region of mtDNA using synthetic RNA guides, we observed a nearly threefold reduction in mtDNA copy number in human cell lines. This proof of concept study demonstrates that a bacterial Argonaute can remain active within the mitochondrial environment and influence mtDNA levels. These findings lay the groundwork for further development of programmable systems for mitochondrial genome manipulation. HighlightsAmAgo targeted to mitochondria significantly reduces human mtDNA copy number Mitochondrial localization of AmAgo is essential for changes in the mtDNA content AmAgo effects on mtDNA are modulated by exogenous guide RNAs Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/689640v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@ddd069org.highwire.dtl.DTLVardef@9db9a3org.highwire.dtl.DTLVardef@1068b02org.highwire.dtl.DTLVardef@904c87_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Targeted deletions in human mitochondrial DNA engineered by Type V CRISPR-Cas12a system

Mutations in mitochondrial DNA (mtDNA) contribute to various neuromuscular diseases, with severity depending on heteroplasmy level when mutant and wild-type mtDNA coexist within the same cell. Developing methods to model mtDNA dysfunction is crucial for experimental therapies. Here, we adapted the Type V CRISPR-AsCas12a system, which recognizes AT-rich PAM sequences, for targeted editing of human mtDNA. We show that AsCas12a effector, fused with a mitochondrial targeting sequence (MTS) from Neurospora crassa ATPase subunit 9, is efficiently addressed into human mitochondria and induces specific mtDNA cleavage in human cells. As a proof-of-concept, we demonstrate that AsCas12a, complexed with two crRNAs targeting distant regions of human mtDNA, introduces specific deletions in mtDNA. For the first time, we provide experimental data proving that a CRISPR system can be used not only for mtDNA degradation but also for precise mtDNA manipulation, offering a potential therapeutic avenue to address mitochondrial disorders.

molecular biology↗

bioRxiv2023

Unravelling the Mitochondrial Mutational Landscape in Chordates: damage-induced versus replication-induced signatures, their Etiologies, and dynamics

To elucidate the primary factors shaping mitochondrial DNA (mtDNA) mutagenesis, we derived a comprehensive 192-component mtDNA mutational spectrum using 86,149 polymorphic synonymous mutations reconstructed from the CytB gene of 967 chordate species. The mtDNA spectrum analysis provided numerous findings on repair and mutation processes, breaking it down into three main signatures: (i) symmetrical, evenly distributed across both strands, mutations, induced by gamma DNA polymerase (about 50% of all mutations); (ii) asymmetrical, heavy-strand-specific, C>T mutations (about 30%); and (iii) asymmetrical, heavy-strand-specific A>G mutations, influenced by metabolic and age-specific factors (about 20%). We propose that both asymmetrical signatures are driven by single-strand specific damage coupled with inefficient base excision repair on the lagging (heavy) strand of mtDNA. Understanding the detailed mechanisms of this damage is crucial for developing strategies to reduce somatic mtDNA mutational load, which is vital for combating age-related diseases.

bioinformatics↗

bioRxiv2022

The mitochondrial component of human infertility- an increased mtDNA content and an excess of ultra-rare mtDNA variants in aneuploid versus euploid embryos.

Using low-coverage, whole-genome sequences of trophectoderm biopsies from 11,610 human blastocyst-stage embryos, we analyzed the relationship between chromosomal abnormalities and mitochondrial (mt) DNA dynamics. Comparing 6,208 aneuploid and 5,402 euploid embryos in cohort studies, we found that mtDNA content in aneuploid embryos was significantly higher than that in euploid embryos. This outcome was confirmed through intrafamilial analyses of embryos with matched parents and in vitro fertilization cycles, and it occurred independent of maternal age. Additional human population-based studies uncovered a higher abundance of ultra-rare mtDNA variants located in never-altered positions in the human population in aneuploid compared to euploid embryos in both cohort- and family-based analyses. This maternal age-independent association of increased mtDNA content and aneuploidy in human embryos may reflect a novel mechanism of purifying selection against potentially deleterious mtDNA variants, which arise from germline or early developmental mtDNA damaging events, that occurs in human embryos prior to implantation.

bioinformatics↗

bioRxiv2021

Mitochondrial mutational spectrum is associated with mammalian longevity: a novel signature of oxidative damage.

The mutational spectrum of the mitochondrial DNA (mtDNA) does not resemble any of the known mutational signatures of the nuclear genome and variation in mtDNA mutational spectra between different organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra is affected by the oxidative damage. Assuming that oxidative damage increases with age, we analyze mtDNA mutagenesis of different species. Analysing (i) dozens thousands of somatic mtDNA mutations in samples of different age (ii) 70053 polymorphic synonymous mtDNA substitutions, reconstructed in 424 mammalian species with different generation length and (iii) synonymous nucleotide content of 650 complete mitochondrial genomes of mammalian species we observed that the frequency of AH>GH substitutions (H - heavy chain notation) is twice higher in species with high versus low generation length making their mtDNA more AH poor and GH rich. Considering that AH>GH substitutions are also sensitive to the time spent single stranded (TSSS) during asynchroniuos mtDNA replication we demonstrated that AH>GH substitution rate is a function of both species-specific generation length and position specific TSSS. We propose that AH>GH is a mitochondria-specific signature of oxidative damage associated with both aging and TSSS.

bioinformatics↗

bioRxiv2020

Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5-scaffold moiety and 3-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

molecular biology↗

bioRxiv2020

A mitochondrial mutational signature of temperature and longevity in ectothermic and endothermic vertebrates.

The variation in the mutational spectrum of the mitochondrial genome (mtDNA) among species is not well understood. Recently, we demonstrated an increase in A>G substitutions on a heavy chain (hereafter AH>GH) of mtDNA in aged mammals, interpreting it as a hallmark of age-related oxidative damage. In this study, we hypothesized that the occurrence of AH>GH substitutions may depend on the level of aerobic metabolism, which can be inferred from an organisms body temperature. To test this hypothesis, we used body temperature in endotherms and environmental temperature in ectotherms as proxies for metabolic rate and reconstructed mtDNA mutational spectra for 1350 vertebrate species. Our results showed that temperature was associated with increased rates of AH>GH and asymmetry of AH>GH in different species of ray-finned fishes and within geographically distinct clades of European anchovy. Analysis of nucleotide composition in the most neutral synonymous sites of fishes revealed that warm-water species were expectedly more A-poor and G-rich compared to cold-water species. Finally, we extended our analyses to all vertebrates and observed higher AH>GH and increased asymmetry of AH>GH in warm-blooded (mammals and birds) compared to cold-blooded (Actinopterygii, amphibia, reptilia) vertebrate classes. Overall, our findings suggest that temperature, through its influence on metabolism and oxidative damage, shapes the mutational properties and nucleotide content of the mtDNA in all vertebrates.

evolutionary biology↗

bioRxiv2019

Deleterious in late life mitochondrial alleles and aging: secrets of Japanese centenarians

Aging in postmitotic tissues is associated with clonal expansion of somatic mitochondrial deletions, the origin of which is not well understood. Deletions in mitochondrial DNA (mtDNA) are often flanked by direct nucleotide repeats, but this alone does not fully explain their distribution. Here, we hypothesized that the close proximity of direct repeats on single-stranded DNA might play a role in the formation of deletions. By analyzing human mtDNA deletions in the major arc of mtDNA, which is single-stranded during replication and is characterized by a high number of deletions, we found a non-uniform distribution with a "hot spot" where one deletion breakpoint occurred within the region of 6-9kb and another within 13-16kb of the mtDNA. This distribution was not explained by the presence of direct repeats, suggesting that other factors, such as the spatial proximity of these two regions can be the cause. In silico analyses revealed that the single-stranded major arc may be organized as a large-scale hairpin-like loop with a center close to 11kb and contacting regions between 6-9 kb and 13-16 kb, which would explain the high deletion activity in this contact zone. The direct repeats located within the contact zone, such as the well-known common repeat with a first arm at 8470-8482 bp and a second arm at 13447-13459 bp, are three times more likely to cause deletions compared to direct repeats located outside of the contact zone. An analysis of age- and disease-associated deletions demonstrated that the contact zone plays a crucial role in explaining the age-associated deletions, emphasizing its importance in the rate of healthy aging. Overall, we provide topological insights into the mechanism of age-associated deletion formation in human mtDNA, which could be used to predict somatic deletion burden and maximum lifespan in different human haplogroups and mammalian species.

genetics↗

bioRxiv2019

Mitochondrial mutational spectrum provides an universal marker of cellular and organismal longevity

Mutational spectrum of the mitochondrial genome (mtDNA) does not resemble signatures of any known mutagens and variation in mtDNA mutational spectra between different tissues and organisms is still incomprehensible. Since mitochondria is tightly involved in aerobic energy production, it is expected that mtDNA mutational spectra may be affected by the oxidative damage which is increasing with cellular and organismal aging. However, the well-documented mutational signature of the oxidative damage, G>T substitutions, is typical only for the nuclear genome while it is extremely rare and age-independent in mtDNA. Thus it is still unclear if there is a mitochondria - specific mutational signature of the oxidative damage. Here, reconstructing mtDNA mutational spectra for human cancers originated from 21 tissues with various cell turnover rate, human oocytes fertilized at different ages, and 424 mammalian species with variable generation length which is a proxy for oocyte age, we observed that the frequency of AH>GH substitutions (H - heavy chain notation) is positively correlated with cellular and organismal longevity. Moreover, this mutational bias from AH to GH affects nucleotide content at the fourfold degenerative synonymous positions leading to a deficit of AH and excess of GH, which is especially pronounced in long-lived mammals. Taking into account additionally, that AH>GH is sensitive to time being single stranded during mtDNA asynchronous replication and A>G is associated with oxidative damage of single-stranded DNA in recent bacterial experiments we propose that AH>GH is a mutational signature of oxidative damage in mtDNA.

genomics↗