Promoter and Terminator Optimization for DNA Methylation Targeting in Arabidopsis

DNA methylation is an important epigenetic mark involved in gene regulation and silencing of transposable elements. The presence or absence of DNA methylation at specific sites can influence nearby gene expression and cause phenotypic changes that remain stable over generations. Recently, development of new technologies has enabled the targeted addition or removal of DNA methylation at specific sites of the genome. Of these new technologies, the targeting of the catalytic domain of Nicotiana tabacum DOMAINS REARRANGED METHYLTRANSFERASE 2 (ntDRM2cd) offers a promising tool for the addition of DNA methylation as it can directly methylate DNA. However, the methylation targeting efficiency of constructs using ntDRM2cd thus far has been relatively low. Previous studies have shown that the use of different promoters or terminators can greatly improve genome-editing efficiencies. In this study, we systematically survey a variety of promoter and terminator combinations to identify optimal combinations to use when targeting the addition of DNA methylation in Arabidopsis thaliana.