Examining the Role of MET1 in the Setaria viridis Methylome with CRISPR/Cas9 Genome Editing
Epigenetic variation in plants, including cytosine DNA methylation, provides a novel area for identification and improvement of crop traits for agricultural purposes. DNA methylation occurs in three nucleotide sequence contexts, CG, CHG, and CHH, where H could be A, C, or T nucleotides, of which CG methylation is the most widespread in the plant genome and occurs at heterochromatic sites, as well as near centromeres. CG methylation is maintained by the enzyme Methyltransferase 1 (MET1) which deposits methyl groups with each round of DNA replication. MET1-knockout studies can help characterize the role of these enzymes in the methylome, allowing for development of precise epigenetic-based methods for crop improvement. However, methylome studies have been limited to the dicot organism Arabidopsis thaliana, and little research exists on the role of MET1 in methylation maintenance in other species, particularly monocot plants. Here, I examine the role of MET1 in Setaria viridis, a model monocot organism for agronomically relevant crops such as rice, wheat, corn, and millet. In this project, I design and build editing reagents to knock out the met1 gene in Setaria viridis. Using PCR, restriction enzyme digestion, and sequencing, I characterize the methylation patterns in the met1 mutant S. viridis genome and compare them to the wild-type S. viridis methylome. Elucidation of these methylation mechanisms contributes to the development and refinement of epigenetics-based methods for improving specific traits in crops for agricultural purposes.