CRISPR-based targeting of DNA methylation in Arabidopsis thaliana by a bacterial CG specific DNA methyltransferase.

CRISPR-based targeted modification of epigenetic marks is an important strategy to regulate the expression of genes and their associated phenotypes in plants and animals. The manipulation of DNA cytosine methylation is particularly attractive in plants since DNA methylation changes are often heritable in subsequent plant generations. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H can be any nucleotide but G), methylation at symmetrical CG sites is the most important for gene silencing, and is also the most efficiently maintained through miotic and meiotic cell divisions. Thus, DNA methylation targeting tools that directly install CG methylation are highly desirable. Here we have developed two CRISPR-based CG specific targeted DNA methylation systems for plants using the DNA methyltransferase MQ1 (SssI) from the bacterium Mollicutes spiroplasma. To reduce off-target effects, we used a variant of the enzyme with reduced catalytic activity. We demonstrate that methylation added by MQ1 is highly target-specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research. This study evaluated two new CRISPR-based tools, MQ1v and SunTag-MQ1v, designed to target de novo DNA methylation in plants using MQ1, a CG-specific bacterial methyltransferase. These tools were targeted to the promoter of the gene FWA using three previously-characterized guide RNAs (Papikian et al. 2019). MQ1v and SunTag-MQ1v were targeted to the FWA promoter in fwa epimutants to test whether they could establish DNA methylation over the promoter and affect FWA expression. Control plants were transformed with dMQ1, a version of MQ1v that has no catalytic activity. MQ1v efficiency was also evaluated in the absence of functional transcriptional (drm1 drm2 background) and post-transcriptional gene silencing (rdr6 background). Transformed plants and their descendants were evaluated by assessing FWA expression (qPCR or RNA-seq), and DNA methylation at the FWA promoter (bisulfite-PCR followed by sequencing, or BS-PCR) and genome-wide (bisulfite sequencing, pr BS-seq). BS-seq and/or BS-PCR was performed for individual transformed plants (T1) and/or their descendants (T2, T3, or T4, corresponding to second-, third-, and fourth-generation descendants of the T1 plants, respectively), as well as appropriate dMQ1, fwa and Col (wild-type) controls. RNA-seq experiments were performed in T4 plants in triplicate.