Inactivation of Three Stacked Genes of Cytosolic Peroxiredoxins by Genome Editing

Abstract

A set of peroxidases detoxifies H₂O₂ and mediates H₂O₂-dependent signal propagation. The peroxidases include peroxiredoxins, glutathione peroxidases, ascorbate peroxidases, and catalases. This at least partial redundancy impedes addressing individual proteins in living plant cells so that the protein functions are often studied by biochemical assays in vitro. In vivo analysis frequently relies on transgenic insertion lines resulting in the knockdown or knockout of the protein of interest. However, many proteins have multiple isoforms in close genomic arrangement so that even crossing of transgenic lines does not allow for a knockdown of all isoforms. The genes encoding for the three cytosolic peroxiredoxins PRXIIB, C, and D in Arabidopsis thaliana are located in close vicinity on chromosome 1 so that crossing over between the genes most rarely occurs and successful crossing of the plants appears impossible. Genome editing instead allows targeting of multiple isoforms and knocks out several genes at once. This chapter describes how to inactivate the three cytosolic peroxiredoxins by CRISPR/Cas9 in A. thaliana.

Keywords: Arabidopsis thaliana; CRISPR/Cas9; Genome editing; Isogenes; Peroxiredoxins.