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

• Published in: Plant science (Limerick) 2025-03, Vol.352, p.112376, Article 112376
• Main Authors: Miao, Wei, Dai, Jie, Zhang, Li, Liang, Zhile, Sun, Xiaoxuan, Huang, Meizi, Zhang, Aqin, Zheng, Long, Li, Yongjun, Li, Ying
• Format: Article
• Language: English
• Subjects: CRISPR; DNA methylation; Gene co-expression; Reduced luminescence 1 (rl1); Reverse genetics
• ISSN: 0168-9452; 1873-2259; 1873-2259
• DOI: 10.1016/j.plantsci.2024.112376

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. •We proposed a novel scheme to obtain low luminescence mutants by reverse genetics.•Experiment results reveal that combining co-expressed gene and CRISPR is feasible.•By this method, we obtained a new mutant (rl1) with reduced LUC luminescence.•rl1 shows genome-wide DNA hypermethylation and can regulate DNA methylation.