Division of Nutritional Sciences, Cornell University, Ithaca, NY, USA.
School of Life Science and Technology, China Pharmaceutical University, Nanjing, China.
Nat Chem Biol. 2019 Sep;15(9):865-871. doi: 10.1038/s41589-019-0327-1. Epub 2019 Aug 5.
RNA modification in the form of N-methyladenosine (mA) regulates nearly all the post-transcriptional processes. The asymmetric mA deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual mA modifications. Here we report the development of 'mA editing', a powerful approach that enables mA installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain mA methyltransferase that can be programmed with a guide RNA. The resultant mA 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered mA 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable mA editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.
RNA 修饰形式的 N6-甲基腺苷（m6A）调控几乎所有的转录后过程。不对称的 m6A 沉积表明区域甲基化可能具有不同的功能后果。然而，目前的 RNA 生物学工具无法区分单个 m6A 修饰的贡献。在这里，我们报告了“m6A 编辑”的发展，这是一种强大的方法，可以在不改变 RNA 一级序列的情况下，在细胞 RNA 中进行 m6A 的安装和擦除。我们设计了 CRISPR-Cas9 和单链 m6A 甲基转移酶的融合蛋白，该融合蛋白可以通过指导 RNA 进行编程。由此产生的 m6A“写入器”允许在不同信使 RNA 区域的单个位点甲基化进行功能比较。我们进一步通过将 CRISPR-Cas9 与 ALKBH5 或 FTO 融合来设计 m6A“擦除器”，以实现 RNA 的特异性去甲基化。可编程 m6A 编辑的发展不仅扩展了 RNA 工程的范围，而且有助于对表观转录组的机制理解。
