title: SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD.
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.
authors.affiliation: Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Cramer-Morales
authors.affiliation: Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242.
authors.forename: Collin D
authors.initials: CD
authors.lastname: Heer
authors.affiliation: Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242.
authors.forename: Kranti A
authors.initials: KA
authors.lastname: Mapuskar
authors.affiliation: Department of Radiation Oncology, B180 Medical Laboratories, The University of Iowa, Iowa City, IA 52242. Electronic address: frederick-domann@uiowa.edu.
authors.forename: Frederick E
authors.initials: FE
authors.lastname: Domann
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D064113:CRISPR-Cas Systems / Q000235:genetics*; D049109:Cell Proliferation; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010085:Oxidative Phosphorylation; D018384:Oxidative Stress; D017393:RNA Editing / Q000235:genetics*; D013385:Succinate Dehydrogenase / Q000378:metabolism; D013482:Superoxide Dismutase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000099156:Superoxide Dismutase 2
chemical_list: D013385:Succinate Dehydrogenase; D013482:Superoxide Dismutase; D000099156:Superoxide Dismutase 2; C491545:SDHD protein, human
keywords: Electron transport; Iron metabolism; Mitochondria; Respiration; Superoxide dismutase