Abstract
CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the 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.
Highlights
CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes
DNA methylation is maintained via three main mechanisms, depending on genomic location and sequence context: CG methylation is maintained by DNA METHYLTRANSFERASE 1 (MET1) [1], non-CG methylation at small transposons and repetitive elements in open chromatin is maintained by RNA-directed DNA methylation (RdDM), and non-CG methylation in dense heterochromatin is maintained by CHROMOMETHYLASE 2 (CMT2) and CHROMOMETHYLASE 3 (CMT3) [3, 4]
We have developed a targeted DNA methylation tool based on a bacterial methyltransferase and the CRISPR-Cas9 platform to directly methylate cytosines at CG sites in Arabidopsis
Summary
CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. The CRISPR-based tools developed in plants have used the catalytic domain of the RdDM methyltransferase DRM2, which adds DNA methylation in all sequence contexts but has a preference for the CHH context [8, 9]. While these tools are able to cause gene silencing, they work at a relatively low efficiency, and the heritability of the targeted DNA methylation in the absence of the effector transgene is often incomplete [8]. We show that this MQ1(Q147L) variant can be used for accurate targeted de novo CG DNA methylation in Arabidopsis
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