Visualizing, quantifying, and manipulating mitochondrial DNA in vivo

Abstract

Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in ageing. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount, and sequence of, mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify and manipulate the properties of mtDNA within cells.

Potential of Mitochondrial Genome Editing for Human Fertility Health

“…Additionally, restriction enzymes, such as EcoRI, PstI, or XhoI, can be targeted to mitochondria used to decrease or eliminate mtDNA in cells. Manipulation of the mtDNA copy number can also be achieved by alteration of the mtDNA replication machinery ( Bacman et al, 2014 ; Prole et al, 2020 ). Overexpression or knockdown of the key component TFAM will increase or decrease the mtDNA number ( Lewis et al, 2016 ; Desdin-Mico et al, 2020 ).…”

“…Moreover, nucleoside analogs, such as the antiretroviral drug zalcitabine that inhibit mitochondrial POLG and cause a decrease in mtDNA copy number, have been used to manipulate mtDNA counts in cells ( Young and Copeland, 2016 ). Moreover, it is predicted that alterations in mitochondrial network fragmentation, which is intimately involved in mechanisms of mitophagy, including Drp1, Opa1, and Mfn1/2, may also enhance the breakdown of mtDNA ( Giacomello et al, 2020 ; Prole et al, 2020 ).…”

“…To date, challenges associated with the delivery of gRNA into the mitochondria have been the major barrier to applying CRISPR/Cas editing technology to mtDNA ( Prole et al, 2020 ). Notably, it was predicted that the combination of modified gRNA with mitochondrial targeting sequence-mediated shuttling of CRISPR/Cas9 proteins may eventually allow precise editing of the mitochondrial genome ( Dan et al, 2021 ).…”

“…Manipulation of mtDNA via MGE could be further divided into the perturbing of mtDNA copy number as well as the repair and elimination of pathogenic mutant mtDNA ( Prole et al, 2020 ). On the one hand, cleavage and breakdown of mtDNA has been achieved using mitochondria-targeted TALENs (mito-TALENs; Bacman et al, 2013 , Yang et al, 2018 ).…”

Modulation of Cellular Fate of Vinyl Triarylamines through Structural Fine Tuning: To Stay or Not To Stay in the Mitochondria?

“…This question in particular is of broad interest as several studies have reported that cationic dyes could target secondary DNA structures in mtDNA but without providing direct evidence [20] . Furthermore finding new labels with a variety of excitation wavelengths for visualizing mtDNA is also of great interest as mentioned in a recent report [21] . Therefore, stimulated by the recent impetus for dye targeting mitochondria, it was decided to address these questions through molecular engineering of the TP‐2Bzim compound (Figure 1), which represents the best candidate of the TPA series in terms of brightness and DNA binding affinity.…”