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

被引:26
作者
Cramer-Morales, Kimberly [1 ]
Heer, Collin D. [1 ]
Mapuskar, Kranti A. [1 ]
Domann, Frederick E. [1 ]
机构
[1] Univ Iowa, Dept Radiat Oncol, Med Labs B180, Iowa City, IA 52242 USA
关键词
Superoxide dismutase; Mitochondria; Respiration; Electron transport; Iron metabolism; MANGANESE-SUPEROXIDE-DISMUTASE; 2 DISTINCT REGIONS; OXIDATIVE STRESS; BREAST-CANCER; MUTANT MICE; ESCHERICHIA-COLI; MESSENGER-RNA; LONG ARM; EXPRESSION; DELETION;
D O I
10.1016/j.freeradbiomed.2015.07.017
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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. (C) 2015 Elsevier Inc. All rights reserved.
引用
收藏
页码:379 / 386
页数:8
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