Department of Chemistry, University at Albany, 1400 Washington Ave, Albany, New York 12222, United States.
Department of Chemistry, Stanford University, 450 Serra Mall, Stanford, California 94305, United States.
ACS Chem Biol. 2024 Aug 16;19(8):1719-1724. doi: 10.1021/acschembio.4c00117. Epub 2024 Jul 25.
The CRISPR-Cas9 system is a widely popular tool for genome engineering. There is strong interest in developing tools for temporal control of CRISPR-Cas9 activity to address some of the challenges and to broaden the scope of potential applications. In this work, we describe a bio-orthogonal chemistry-based approach to control nuclease activity with temporal precision. We report a -cyclooctene (TCO)-acylimidazole reagent that acylates 2'-OH groups of RNA. Poly acylation ("cloaking") of RNA was optimized using a model 18-nt oligonucleotide, as well as CRISPR single guide RNA (sgRNA). Two hours of treatment completely inactivated sgRNA for Cas9-assisted DNA cleavage. Nuclease activity was restored upon addition of tetrazine, which removes the TCO moieties via a two-step process ("uncloaking"). The approach was applied to target the GFP gene in live HEK293 cells. GFP expression was analyzed by flow cytometry. In the future, we anticipate that our approach will be useful in the field of developmental biology, by enabling investigation of genes of interest at different stages of an organism's development.
CRISPR-Cas9 系统是一种广泛应用的基因组工程工具。人们强烈希望开发出用于 CRISPR-Cas9 活性的时间控制工具，以解决一些挑战并拓宽潜在应用的范围。在这项工作中，我们描述了一种基于生物正交化学的方法，可实现具有时间精度的核酸酶活性控制。我们报告了一种 -环辛烯 (TCO)-酰亚咪唑试剂，它酰化 RNA 的 2'-OH 基团。通过使用模型 18 个核苷酸的寡核苷酸以及 CRISPR 单指导 RNA (sgRNA) 优化了 RNA 的多酰化（“伪装”）。两小时的处理可完全使 sgRNA 失活，从而阻止 Cas9 辅助的 DNA 切割。加入四嗪后可恢复核酸酶活性，四嗪通过两步过程（“解伪装”）去除 TCO 部分。该方法应用于靶向活 HEK293 细胞中的 GFP 基因。通过流式细胞术分析 GFP 表达。将来，我们预计我们的方法将在发育生物学领域非常有用，可用于在生物体发育的不同阶段研究感兴趣的基因。
