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.

 摘 要 

基于CRISPR的表观标记靶向修饰，比如DNA胞嘧啶甲基化，是调控基因表达及其相关表型的重要策略。尽管植物具有多种不同类型的DNA甲基化类型，即CG、CHG和CHH（其中，H为A、T或C），但对称性的CG甲基化对于基因沉默尤其重要，并且在有丝分裂和减数分裂时期，CG类型的甲基化能够很好的维持住甲基化状态。因此，可以设想开发出一种可以直接将CG甲基化添加到基因组上特定位点的工具具有非常重要的意义，但目前在植物中仍缺乏这一类的工具。本文中，作者开发了两个适用于植物的基于CRISPR的CG特异性靶向DNA甲基化系统，该系统基于一种活性降低但特异性高的细菌CG特异性DNA甲基转移酶MQ1变体。作者的实验表明，MQ1的甲基化具有高度的靶向特异性，并且可以在没有效应子的情况下遗传维持。本文所开发的工具在作物工程和植物遗传研究中具有非常重要的价值。