A new method for identifying proteins involved in DNA methylation through reverse genetics in Arabidopsis.

Forward genetic screens have uncovered numerous genes involved in DNA methylation regulation, but these methods are often time-intensive, costly, and labor-intensive. To address these limitations, this study utilized CRISPR technology to knockout selected co-expressed genes, enabling the rapid identification of low luciferase (LUC) luminescence mutants in the Col-LUC line, which harbors a LUC transgene driven by a 2×35S promoter in Arabidopsis. As proof of concept, the repressor of silencing 1 (ROS1) and RNA-directed DNA methylation 1 (RDM1) genes were used as controls, while the increased DNA methylation 3 (IDM3) gene, co-expressed with ROS1, was selected as the target for gene knockout experiments. The results demonstrated that combining co-expression analysis with CRISPR technology is an effective strategy for generating low LUC luminescence mutants in the Col-LUC line. Notably, a new mutant, named reduced luminescence 1 (rl1), was identified through this approach. The rl1 mutant exhibited genome-wide DNA hypermethylation, and its reduced luminescence phenotype was largely reversed by treatment with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine, confirming its anti-silencing role in DNA methylation regulation. This study presents a novel and efficient approach for obtaining low luminescence mutants in the Col-LUC line and identifies RL1 as a previously uncharacterized protein involved in DNA methylation regulation.