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How gene editing can address mitochondrial DNA?

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Antonio Carusillo
Antonio Carusillo Aug 27, 2020
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Editing mitochondrial DNA: how can we edit mtDNA and how this may help to understand and cure mitochondrial related disorders?

Mitochondria are key organelles of the cells sustaining its biochemical activity and play also a key role in aging and have been also involved in aging related disorders
The human mitochondrial DNA (mtDNA) is a double-stranded, circular molecule of 16 569 bp and contains 37 genes coding for two rRNAs, 22 tRNAs and 13 polypeptides.

Question of this Session:

Is it possible to tackle this disorders at their very source? Meaning the DNA sequence?

What are the current tools to edit the DNA

In the past decades, gene editing field has seen an unprecedented development due to CRISPR/Cas9. These novel molecular scissors, known also as designer nucleases, allows for precise manipulation of DNA sequence. The system relies on a two components: the Cas9 nuclease which is in charge of cutting the DNA and the guide RNA (gRNA) whose role is to direct the Cas9 nuclease toward the desired targets. By reprogramming the guide RNA sequence it is therefore possible to targeted -ideally- any desired DNA sequence within the genome.

Editing mithocondrial DNA is challenging
Gene editing has made huge progress over the past decades, its efficiency and safety has been improved and the first human clinal trials are currently on going .
However, although the most recent advances, the mtDNA was beyond genetic engineering reach. This is due to the fact that to date the trafficking of Nucleic acids, RNA in particular, has remained elusive.
Based on what we said before, CRISPR/Cas9 technology relies on two main components the Cas9 nuclease and the guide RNA (gRNA) to target the desired DNA sequence. With a poorly efficient trafficking of the gRNA to the mitochondria, CRISPR can not properly work.

How can we tackle this?
- Are there already DNA editing technologies that do not rely on RNA?
- Can we exploit some of the few clues about RNA trafficking to devise a way to import the desired RNA into the mithocondria ? For example based on this recent paper

[1]Sun N, Youle RJ, Finkel T. The Mitochondrial Basis of Aging. Mol Cell. 2016;61(5):654-666. doi:10.1016/j.molcel.2016.01.028

[2]Heidi Chial, Ph.D. (Write Science Right) & Joanna Craig, Ph.D. (Write Science Right) © 2008 Nature Education Citation: Chial, H. & Craig, J. (2008) mtDNA and mitochondrial diseases. Nature Education 1(1):217


[4]Hirakawa MP, Krishnakumar R, Timlin JA, Carney JP, Butler KS. Gene editing and CRISPR in the clinic: current and future perspectives. Biosci Rep. 2020;40(4):BSR20200127. doi:10.1042/BSR20200127

[5]Gammage PA, Moraes CT, Minczuk M. Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet. 2018;34(2):101-110. doi:10.1016/j.tig.2017.11.001

Creative contributions

Using TALEs and Base editing to edit mtDNA

Antonio Carusillo
Antonio Carusillo Aug 28, 2020
Dr. David Liu has developed and improved the first base editors. By using a Cas9 variant fused to specific enzymes, it is possible to convert a C•G base pair into a T•A base pair or A•T base pair to a G•C base pair. This way it is possible to achieve the four transition mutations (C→T, G→A, A→G, and T→C) - which can be applied for genetic Knock Out (you can imagine to introduce a stop codon ) or to edit point mutation causing the phenotype ( for example an aberrant stop codon or splice site like it happens in some immunodeficiencies). This technology was already a real breakthrough and if you just write base editors on PubMed you will get roughly 300 papers and considering that it was developed like 3 years ago it is stunning (1) For what said before, a technologo relying on RNA can not be used. For this reason, David Liu and its team turned to a previous technologies known as transcription activator-like effector (TALEs) whose target recognition is encoded by an arrays of highly conserved 33–35 amino acid repeats flanked by additional TALE-derived domains at the amino- and carboxy-terminal ends of the array (2) Without digging to much into details, the key point is that the TALEs do not require an RNA to be guided towards the target. Thus, they may serve the scope. At this point, the team wanted to combine this with base editing. They used a bacterial toxin - a cytidine deaminase enzyme called DddA - which can convert the cytosine (C) to uracil (U). An important characteristic of DddA is that it targets double-stranded DNA, whereas the cytidine deaminases mentioned above target single-stranded DNA. This is important, cause TALEs upon binding do not stretch open the DNA like CRISPR, hereby do not unwind the DNA to single strand. So, by fusing TALEs to DddA they could apply base editing, and in general genetic engineering, for the very first time to mtDNA (3). Mutations in mtDNA are associated to metabolic conditions as well as neurodegenerative disease (4) On the one hand it is hard to recreate disease model harboring mutation in the mtDNA and on the other it is even harder to address this kind of conditions. The David Liu`s lab may have provided the scientific community with a novel tool to investigate mtDNA related disease as well as with a possible technology to cure those conditions in a near future. References: 1- Rees HA, Liu DR. Base editing: precision chemistry on the genome and transcriptome of living cells [published correction appears in Nat Rev Genet. 2018 Oct 19;:]. Nat Rev Genet. 2018;19(12):770-788. doi:10.1038/s41576-018-0059-1 2- Joung JK, Sander JD. TALENs: a widely applicable technology for targeted genome editing. Nat Rev Mol Cell Biol. 2013;14(1):49-55. doi:10.1038/nrm3486 3-Mok, B. Y. et al. Nature https://10.1038/s41586-020-2477-4 (2020) 4-Cha MY, Kim DK, Mook-Jung I. The role of mitochondrial DNA mutation on neurodegenerative diseases. Exp Mol Med. 2015;47(3):e150. Published 2015 Mar 13. doi:10.1038/emm.2014.122
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Delivering the gRNA expression cassette as a self-replicating plasmid

Manel Lladó Santaeularia
Manel Lladó Santaeularia Dec 06, 2020
As mentioned in the session, the main limitation for the use of Cas9 in mitochondria is not that Cas9 has difficulty entering the mitocondria, but rather the limitation of gRNA entry. Mitochondria very rarely allow entry of DNA and especially RNA, as a mean of protection from viral infections. For this reason, having the gRNA be produced in the nucleus and then enter the mitochondria is not a desirable approach. However, researchers have found methods of delivering DNA to mitochondria that could be exploited to solve this issue. One of these approaches is biolistic microprojectile transformation . As described by the authors, this method "uses a helium shock wave in an evacuated chamber to accelerate microscopic metal particles coated with DNA towards a lawn of cells on a Petri plate. The shockwave is generated by rupture of a membrane at high pressure, and accelerates a second membrane (the macrocarrier or flying disk), carrying the metal particles, towards the plate. Some cells on the plate are penetrated by particles and survive. DNA precipitated on the particles is thus introduced into cells and is readily taken up by the nucleus. In addition, the mitochondria of a small fraction of such transformants also take up DNA". While this method seems at least debatable, Bonnefoy et al. demonstrated that it successfully delivers DNA to mitochondria. The authors generated plasmids containing some mitochondrial sequences and demonstrated that these plasmids are mantained inside mitochondria and have the ability to self-replicate.

Recently, Yoo et al. have demonstrated that this method could be used to deliver a plasmid encoding for Cas9, the gRNA expression cassette and even a donor DNA. This method achieved targeted integration of the donor DNA through homology-directed repair in the mitochondrial DNA of yeast . However, no demonstration of gRNA expression from the Edit plasmids was reported. Although this approach seems interesting, two main limitations seem to arise:
  • Can we apply this in vitro delivery method to human cells? Or at least can we find a similar approach that allows us to deliver self-replicating plasmid DNA to our mitochondria? No such thing has been published so far, but I'm sure this issue will be addressed in the future, maybe with adaptations of technologies such as MITO-Porter

  • Is expression of Cas9 going to be constant in transformed mitochondria? Could that lead to off-target effects that could deplete mitochondrial DNA? In that case, maybe it would be interesting to deliver Cas9 as a protein, to avoid its long-term expression, while delivering the gRNA and potential donor DNA as a plasmid. However, there are yet no reports of such a strategy being effective for mitochondrial gene editing.

  • Is this going to be a better approach than just using TALENs of Base Editing?

[1]Bonnefoy N, Fox TD. Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination. Methods Mol Biol. 2007;372:153-166. doi:10.1007/978-1-59745-365-3_11

[2]Yoo BC, Yadav NS, Orozco EM Jr, Sakai H. Cas9/gRNA-mediated genome editing of yeast mitochondria and Chlamydomonas chloroplasts. PeerJ. 2020;8:e8362. Published 2020 Jan 6. doi:10.7717/peerj.8362

[3]Yamada Y, Akita H, Kamiya H, et al. MITO-Porter: A liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion. Biochim Biophys Acta. 2008;1778(2):423-432. doi:10.1016/j.bbamem.2007.11.002

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Antonio Carusillo
Antonio Carusillo3 years ago
Hi Manel I really like the idea. Probably In cell lines it will be possible, not quite sure in primary cells as they do not really well tolerate plasmid DNA, maybe mini-circles may come handy to this regard. Also would be interesting to see how it works for the Cas9 as protein while the gRNA is a plasmid. I do not know if there are any studies where they tried to do it. Cause I imagine the Cas9 protein being slowly degradated while the gRNA plasmid is still being transcribed. But if you can make it this will also open a new avenue about mtDNA off-target assays!
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