Cold Spring Harbor Laboratory; Faculty of Pharmacy, University of Ljubljana;
Cold Spring Harbor Laboratory.
J Vis Exp. 2021 Sep 2(175). doi: 10.3791/60685.
The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory tool to introduce accurate and targeted modifications in the genome. Its enormous popularity and rapid spread are attributed to its easy use and accuracy compared to its predecessors. Yet, the constitutive activation of the system has limited applications. In this paper, we describe a new method that allows temporal control of CRISPR/Cas9 activity based on conditional stabilization of the Cas9 protein. Fusing an engineered mutant of the rapamycin-binding protein FKBP12 to Cas9 (DD-Cas9) enables the rapid degradation of Cas9 that in turn can be stabilized by the presence of an FKBP12 synthetic ligand (Shield-1). Unlike other inducible methods, this system can be adapted easily to generate bi-cistronic systems to co-express DD-Cas9 with another gene of interest, without conditional regulation of the second gene. This method enables the generation of traceable systems as well as the parallel, independent manipulation of alleles targeted by Cas9 nuclease. The platform of this method can be used for the systematic identification and characterization of essential genes and the interrogation of the functional interactions of genes in in vitro and in vivo settings.
簇状规律间隔短回文重复序列-（CRISPR-）相关蛋白 9（CRISPR/Cas9）技术已成为在基因组中引入精确和靶向修饰的流行实验室工具。与前代技术相比，其易于使用和准确性使其非常受欢迎且迅速普及。然而，该系统的组成性激活限制了其应用。在本文中，我们描述了一种新方法，该方法基于 Cas9 蛋白的条件稳定性来实现 CRISPR/Cas9 活性的时间控制。将雷帕霉素结合蛋白 FKBP12 的工程突变体融合到 Cas9（DD-Cas9）上，可实现 Cas9 的快速降解，而 FKBP12 的合成配体（Shield-1）的存在又可稳定 Cas9。与其他诱导型方法不同，该系统可以轻松适应生成双顺反子系统，以与另一个感兴趣的基因共表达 DD-Cas9，而无需对第二个基因进行条件调节。该方法可用于生成可追踪的系统，以及平行、独立地操纵 Cas9 核酸酶靶向的等位基因。该方法的平台可用于在体外和体内环境中对必需基因进行系统鉴定和特征分析，以及对基因功能相互作用进行分析。
