Genetic Manipulation of Reactive Oxygen Species (ROS) Homeostasis Utilizing CRISPR/Cas9-Based Gene Editing in Rice.

Abstract

Reactive oxygen species (ROS) are now recognized as key signals in plant stress responses. Adverse environmental conditions can either promote ROS production or downregulate antioxidative enzymes, leading to the alteration of redox homeostasis and activation of ROS-linked stress signaling. To uncover their signaling mechanisms and to characterize related components, genetic modification of ROS homeostasis is a central approach. CRISPR/Cas9-based genome editing system has become a powerful tool for gene mutation in a variety of organisms, including plants. Within this chapter, we describe a method that can be applied to manipulate ROS homeostasis in rice (Oryza sativa L.) utilizing CRISPR/Cas9 technology. Step-by-step protocols including the design and construction of Cas9/sgRNA, agrobacterium-mediated transformation, and mutation characterization are described. Application of this system in editing a rice catalase gene CatC, a key antioxidative enzyme in controlling ROS homeostasis, is also presented.