Basudev Ghoshal,  Colette L. Picard, Brandon Vong, Suhua Feng, and Steven E. Jaco

PNAS June 8, 2021 118 (23) e2125016118

Significance

Site-specific modification of epigenetic marks such as DNA methylation to regulate gene expression is a unique approach to enhance economically important crop traits. This approach allows for the maintenance of the introduced modifications in the absence of the initial transgene inducer in subsequent generations and relies largely on methylation of cytosines in the CG-specific sequence context. 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. These tools expand the currently available CRISPR-based targeted DNA methylation tools and provide an approach for the establishment of heritable targeted DNA methylation in plants.

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.

See: https://www.pnas.org/content/118/23/e2125016118

Figure 1: MQ1v targets DNA methylation to FWA and causes an early-flowering phenotype. (A) Bisulfite PCR sequencing of two T1 lines (T1-3, T1-7) transformed with MQ1v and a negative control (T1-1) transformed with dMQ1, over three regions of the FWA promoter region: region 1 (Chr4:13038143–13038272), region 2 (Chr4:13038356–13038499), and region 3 (Chr4:13038568–13038695). Each T1 plant is a result of an independent transgenic event. (B) Stacked bar plot showing the percentage of T2 early- and late-flowering plants in the progeny of two different T1 lines (T1-3 and T1-7), together with wild-type Col, and T2 progenies of a matched dMQ1 (T1-1) control. Percentage of early-flowering plants is denoted in the bar plots. The total numbers (N) of plants assayed for each line were 3, 13, 17, and 32. (C) DNA methylation profile at the FWA promoter region in early-flowering progeny of two T1 plants (T2-3-a, T2-3-b are progeny of T2-3; T2-7-a, T2-7-b are progeny of T2-7), along with Col and a matched dMQ1 (T2-1-a) plant as controls. Only cytosines with at least five overlapping reads are shown. Small, negative values (black) indicate cytosines with five or more overlapping reads but no DNA methylation. Vertical yellow bars indicate the locations of the guide 4, guide 10, and guide 18 binding sites.