SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

Kimberly Cramer-Morales; Collin D Heer; Kranti A Mapuskar; Frederick E Domann

doi:10.1016/j.freeradbiomed.2015.07.017

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SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

Journal article

Open access

Peer reviewed

SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD

Kimberly Cramer-Morales, Collin D Heer, Kranti A Mapuskar and Frederick E Domann

Free radical biology & medicine, Vol.89, pp.379-386

12/2015

DOI: 10.1016/j.freeradbiomed.2015.07.017

PMCID: PMC4890619

PMID: 26208779

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http://doi.org/10.1016/j.freeradbiomed.2015.07.017View

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Abstract

To date no models exist to study MnSOD deficiency in human cells. To address this deficiency, we created a SOD2-null human cell line that is completely devoid of detectable MnSOD protein expression and enzyme activity. We utilized the CRISPR/Cas9 system to generate biallelic SOD2 disruption in HEK293T cells. These SOD2-null cells exhibit impaired clonogenic activity, which was rescued by either treatment with GC4419, a pharmacological small-molecule mimic of SOD, or growth in hypoxia. The phenotype of these cells is primarily characterized by impaired mitochondrial bioenergetics. The SOD2-null cells displayed perturbations in their mitochondrial ultrastructure and preferred glycolysis as opposed to oxidative phosphorylation to generate ATP. The activities of mitochondrial complex I and II were both significantly impaired by the absence of MnSOD activity, presumably from disruption of the Fe/S centers in NADH dehydrogenase and succinate dehydrogenase subunit B by the aberrant redox state in the mitochondrial matrix of SOD2-null cells. By creating this model we provide a novel tool with which to study the consequences of lack of MnSOD activity in human cells.

Superoxide Dismutase - genetics

Cell Proliferation

Oxidation-Reduction

Oxidative Stress

RNA Editing - genetics

Humans

Gene Expression Regulation

Oxidative Phosphorylation

Molecular Sequence Data

Superoxide Dismutase - deficiency

Mitochondria - metabolism

Mitochondria - pathology

CRISPR-Cas Systems - genetics

Blotting, Western

Base Sequence

HEK293 Cells

Succinate Dehydrogenase - metabolism

Superoxide Dismutase - metabolism

Details

Title: Subtitle: SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD
Creators: Kimberly Cramer-Morales - Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242
Collin D Heer - Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242
Kranti A Mapuskar - Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242
Frederick E Domann - Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242. Electronic address: frederick-domann@uiowa.edu
Resource Type: Journal article
Publication Details: Free radical biology & medicine, Vol.89, pp.379-386
DOI: 10.1016/j.freeradbiomed.2015.07.017
PMID: 26208779
PMCID: PMC4890619
NLM abbreviation: Free Radic Biol Med
ISSN: 0891-5849
eISSN: 1873-4596
Publisher: United States
Grant note: P30 ES005605 / NIEHS NIH HHS P30 CA086862 / NCI NIH HHS T32 CA078586 / NCI NIH HHS
Language: English
Date published: 12/2015
Academic Unit: Pathology; Surgery; Radiation Oncology
Record Identifier: 9984047613302771

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