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Site-specific CRISPR-based mitochondrial DNA manipulation is limited by gRNA import

Schmiderer, Ludwig LU ; Yudovich, David LU ; Oburoglu, Leal LU orcid ; Hjort, Martin LU orcid and Larsson, Jonas LU (2022) In Scientific Reports 12. p.1-9
Abstract

Achieving CRISPR Cas9-based manipulation of mitochondrial DNA (mtDNA) has been a long-standing goal and would be of great relevance for disease modeling and for clinical applications. In this project, we aimed to deliver Cas9 into the mitochondria of human cells and analyzed Cas9-induced mtDNA cleavage and measured the resulting mtDNA depletion with multiplexed qPCR. In initial experiments, we found that measuring subtle effects on mtDNA copy numbers is challenging because of high biological variability, and detected no significant Cas9-caused mtDNA degradation. To overcome the challenge of being able to detect Cas9 activity on mtDNA, we delivered cytosine base editor Cas9-BE3 to mitochondria and measured its effect (C → T mutations) on... (More)

Achieving CRISPR Cas9-based manipulation of mitochondrial DNA (mtDNA) has been a long-standing goal and would be of great relevance for disease modeling and for clinical applications. In this project, we aimed to deliver Cas9 into the mitochondria of human cells and analyzed Cas9-induced mtDNA cleavage and measured the resulting mtDNA depletion with multiplexed qPCR. In initial experiments, we found that measuring subtle effects on mtDNA copy numbers is challenging because of high biological variability, and detected no significant Cas9-caused mtDNA degradation. To overcome the challenge of being able to detect Cas9 activity on mtDNA, we delivered cytosine base editor Cas9-BE3 to mitochondria and measured its effect (C → T mutations) on mtDNA. Unlike regular Cas9-cutting, this leaves a permanent mark on mtDNA that can be detected with amplicon sequencing, even if the efficiency is low. We detected low levels of C → T mutations in cells that were exposed to mitochondrially targeted Cas9-BE3, but, surprisingly, these occurred regardless of whether a guide RNA (gRNA) specific to the targeted site, or non-targeting gRNA was used. This unspecific off-target activity shows that Cas9-BE3 can technically edit mtDNA, but also strongly indicates that gRNA import to mitochondria was not successful. Going forward mitochondria-targeted Cas9 base editors will be a useful tool for validating successful gRNA delivery to mitochondria without the ambiguity of approaches that rely on quantifying mtDNA copy numbers.

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author
; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Scientific Reports
volume
12
article number
18687
pages
1 - 9
publisher
Nature Publishing Group
external identifiers
  • scopus:85141140699
  • pmid:36333335
ISSN
2045-2322
DOI
10.1038/s41598-022-21794-0
language
English
LU publication?
yes
additional info
Funding Information: We are grateful to Emanuela Monni for technical assistance and help with confocal microscopy. We are thankful for support of the Lund Stem Cell Center FACS Facility. We thank David Liu for providing the pCMV-BE3 plasmid, Feng Zhang for providing lentiCRISPR v2 plasmid, and Antal Kiss and Paul Modrich for providing pAN4 plasmid. We thank Joseph Cardiello for critical reading of the manuscript. Publisher Copyright: © 2022, The Author(s).
id
6a053238-87c5-41c1-a95a-ea086cd533e1
date added to LUP
2022-12-05 08:32:16
date last changed
2024-06-14 23:35:09
@article{6a053238-87c5-41c1-a95a-ea086cd533e1,
  abstract     = {{<p>Achieving CRISPR Cas9-based manipulation of mitochondrial DNA (mtDNA) has been a long-standing goal and would be of great relevance for disease modeling and for clinical applications. In this project, we aimed to deliver Cas9 into the mitochondria of human cells and analyzed Cas9-induced mtDNA cleavage and measured the resulting mtDNA depletion with multiplexed qPCR. In initial experiments, we found that measuring subtle effects on mtDNA copy numbers is challenging because of high biological variability, and detected no significant Cas9-caused mtDNA degradation. To overcome the challenge of being able to detect Cas9 activity on mtDNA, we delivered cytosine base editor Cas9-BE3 to mitochondria and measured its effect (C → T mutations) on mtDNA. Unlike regular Cas9-cutting, this leaves a permanent mark on mtDNA that can be detected with amplicon sequencing, even if the efficiency is low. We detected low levels of C → T mutations in cells that were exposed to mitochondrially targeted Cas9-BE3, but, surprisingly, these occurred regardless of whether a guide RNA (gRNA) specific to the targeted site, or non-targeting gRNA was used. This unspecific off-target activity shows that Cas9-BE3 can technically edit mtDNA, but also strongly indicates that gRNA import to mitochondria was not successful. Going forward mitochondria-targeted Cas9 base editors will be a useful tool for validating successful gRNA delivery to mitochondria without the ambiguity of approaches that rely on quantifying mtDNA copy numbers.</p>}},
  author       = {{Schmiderer, Ludwig and Yudovich, David and Oburoglu, Leal and Hjort, Martin and Larsson, Jonas}},
  issn         = {{2045-2322}},
  language     = {{eng}},
  pages        = {{1--9}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Scientific Reports}},
  title        = {{Site-specific CRISPR-based mitochondrial DNA manipulation is limited by gRNA import}},
  url          = {{http://dx.doi.org/10.1038/s41598-022-21794-0}},
  doi          = {{10.1038/s41598-022-21794-0}},
  volume       = {{12}},
  year         = {{2022}},
}