CRISPR-dCas9-Based Targeted Manipulation of DNA Methylation in Plants

DNA cytosine methylation is an important epigenetic mark involved in the regulation of gene expression, transposon silencing, and genome integrity. In plants, targeted manipulation of DNA methylation is achieved via artificial zinc finger (ZF) or clustered regularly interspaced short palindromic repeats (CRISPR)-based tools. These tools can recruit various effectors, which can catalyze either the addition or removal of DNA methylation at a specific locus. The relatively low cost and ease of targeting specific regions within the genome by CRISPR-based systems make these tools an attractive option for targeted manipulation of DNA methylation. Here, we provide a detailed protocol for generating CRISPR-based constructs designed to add or remove DNA methylation in plants in a sequence-specific manner, using the SunTag system. We also discuss methods for analyzing DNA methylation data to validate the effectiveness and specificity of the construct. Our protocol will make these tools accessible to individuals interested in targeted manipulation of DNA methylation in plants for basic research or crop engineering.