CRISPR/Cas9-Targeted Disruption of Two Highly Homologous Arabidopsis thaliana DSS1 Genes with Roles in Development and the Oxidative Stress Response

Global climate change has a detrimental effect on plant growth and health, causing serious losses in agriculture. Investigation of the molecular mechanisms of plant responses to various environmental pressures and the generation of plants tolerant to abiotic stress are imperative to modern plant science. In this paper, we focus on the application of the well-established technology CRISPR/Cas9 genome editing to better understand the functioning of the intrinsically disordered protein DSS1 in plant response to oxidative stress. The Arabidopsis genome contains two highly homologous DSS1 genes, AtDSS1(I) and AtDSS1(V). This study was designed to identify the functional differences between AtDSS1s, focusing on their potential roles in oxidative stress. We generated single dss1(I) and dss1(V) mutant lines of both Arabidopsis DSS1 genes using CRISPR/Cas9 technology. The homozygous mutant lines with large indels (dss1(I)del25 and dss1(V)ins18) were phenotypically characterized during plant development and their sensitivity to oxidative stress was analyzed. The characterization of mutant lines revealed differences in root and stem lengths, and rosette area size. Plants with a disrupted AtDSS1(V) gene exhibited lower survival rates and increased levels of oxidized proteins in comparison to WT plants exposed to oxidative stress induced by hydrogen peroxide. In this work, the dss1 double mutant was not obtained due to embryonic lethality. These results suggest that the DSS1(V) protein could be an important molecular component in plant abiotic stress response.