CRISPR/Cas9-mediated knockout of p22phox leads to loss of Nox1 and Nox4, but not Nox5 activity

  • The NADPH oxidases are important transmembrane proteins producing reactive oxygen species (ROS). Within the Nox family, different modes of activation can be discriminated. Nox1-3 are dependent on different cytosolic subunits, Nox4 seems to be constitutively active and Nox5 is directly activated by calcium. With the exception of Nox5, all Nox family members are thought to depend on the small transmembrane protein p22phox. With the discovery of the CRISPR/Cas9-system, a tool to alter genomic DNA sequences has become available. So far, this method has not been widely used in the redox community. On such basis, we decided to study the requirement of p22phox in the Nox complex using CRISPR/Cas9-mediated knockout. Knockout of the gene of p22phox, CYBA, led to an ablation of activity of Nox4 and Nox1 but not of Nox5. Production of hydrogen peroxide or superoxide after knockout could be rescued with either human or rat p22phox, but not with the DUOX-maturation factors DUOXA1/A2. Furthermore, different mutations of p22phox were studied regarding the influence on Nox4-dependent H2O2 production. P22phox Q130* and Y121H affected maturation and activity of Nox4. Hence, Nox5-dependent O2•- production is independent of p22phox, but native p22phox is needed for maturation of Nox4 and production of H2O2.

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Author:Kim-Kristin Prior, Matthias LeisegangORCiDGND, Ivana Josipovic, Oliver Löwe, Ajay M. ShahORCiD, Norbert WeißmannGND, Katrin SchröderORCiDGND, Ralf BrandesORCiDGND
Pubmed Id:
Parent Title (English):Redox Biology
Place of publication:Amsterdam [u.a.]
Document Type:Article
Year of Completion:2016
Date of first Publication:2016/08/24
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2016/11/01
Tag:CRISPR/Cas9; CYBA/p22phox; NADPH oxidase
Page Number:9
First Page:287
Last Page:295
(c) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND-license (
Institutes:Medizin / Medizin
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
Licence (German):License LogoCreative Commons - Namensnennung-Nicht kommerziell - Keine Bearbeitung 4.0