CELLECTIS S.A., 8 Rue de la Croix Jarry, 75013 Paris, France.
J Biol Chem. 2012 Aug 31;287(36):30139-50. doi: 10.1074/jbc.M112.379966. Epub 2012 Jun 27.
In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.
在这项研究中，我们探讨了 CpG 甲基化是否会影响用于基因组工程应用的巨型核酸酶的 DNA 结合亲和力和活性。生化和结构方法的结合使我们能够证明 CpG 甲基化降低了 I-CreI 的 DNA 结合亲和力，并抑制了其在体外的内切酶活性。这种抑制取决于甲基化胞嘧啶在 DNA 靶标的位置，当它位于中央四碱基内时，抑制几乎是完全的。I-CreI 与甲基化同源靶 DNA 结合的晶体结构为这种抑制提供了分子基础，尽管确切的机制仍有待确定。最后，我们证明了靶向内源性位点的 CpG 甲基化会降低工程巨型核酸酶的功效，我们提出了一种合理的巨型核酸酶 DNA 结合域设计，以减轻这种影响。我们的结论是，尽管工程巨型核酸酶的活性和序列特异性是至关重要的参数，但为了成功进行基因编辑，需要考虑目标 DNA 的表观遗传修饰。
