Department of Chemistry, Case Western Reserve University, 2080 Adelbert Rd, Cleveland, OH 44106, USA.
Angew Chem Int Ed Engl. 2023 Oct 23;62(43):e202309291. doi: 10.1002/anie.202309291. Epub 2023 Sep 15.
N6-methyladenosine (m A) on RNAs plays an important role in regulating various biological processes and CRIPSR technology has been employed for programmable m A editing. However, the bulky size of CRISPR protein and constitutively expressed CRISPR/RNA editing enzymes can interfere with the native function of target RNAs and cells. Herein, we reported a conditional m A editing platform (FKBP*-dCas13b-ALK) based on a ligand stabilized dCas13 editor. The inducible expression of this m A editing system was achieved by adding or removing the Shield-1 molecule. We further demonstrated that the targeted recruitment of dCas13b-m A eraser fusion protein and site-specific m A erasing were achieved under the control of Shield-1. Moreover, the release and degradation of dCas13b fusion protein occurred faster than the restoration of m A on the target RNAs after Shield-1 removal, which provides an ideal opportunity to study the m A function with minimal steric interference from bulky dCas13b fusion protein.
N6-甲基腺苷（m A）在 RNA 上发挥着重要作用，调控着各种生物过程，CRIPSR 技术已被用于可编程 m A 编辑。然而，CRISPR 蛋白的庞大体积和组成型表达的 CRISPR/RNA 编辑酶会干扰靶 RNA 和细胞的固有功能。在此，我们报道了一种基于配体稳定的 dCas13 编辑器的条件性 m A 编辑平台（FKBP*-dCas13b-ALK）。通过添加或去除 Shield-1 分子，实现了该 m A 编辑系统的诱导表达。我们进一步证明，在 Shield-1 的控制下，靶向募集 dCas13b-m A 擦除融合蛋白和靶向 m A 擦除得以实现。此外，dCas13b 融合蛋白的释放和降解比 Shield-1 去除后靶 RNA 上 m A 的恢复更快，这为研究 m A 功能提供了一个理想的机会，可最大限度地减少庞大的 dCas13b 融合蛋白的空间干扰。
