Department of Physiology & Biophysics, University of Washington School of Medicine, Seattle, Washington.
Bieler School of Environment, McGill University, Montreal, Quebec, Canada.
Curr Protoc. 2023 Dec;3(12):e965. doi: 10.1002/cpz1.965.
Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin-relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off-target effects are minimized by testing for the most efficient target site using a split-GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Choosing a target site for gene modification Basic Protocol 2: Design of gRNA(s) for targeted gene modification Basic Protocol 3: Split-GFP test for target efficiency Basic Protocol 4: Design of the recombination template and analytical primers Support Protocol 1: Design of primers for analytical PCR Basic Protocol 5: Transfection, isolation, and validation of engineered cells Support Protocol 2: Stable transfection of engineered cells with binding partners.
蛋白质的活性通常在功能上是整合的，并局限于细胞内的关键位置。Knocksideways 实验允许研究人员快速将蛋白质转移到细胞的替代或异位区域，并评估由此产生的细胞反应。简而言之，使用这种方法测试的单个蛋白质必须用在雷帕霉素处理下二聚化的部分进行修饰，以促进实验的空间重定位。CRISPR 技术使研究人员能够在细胞中直接对修饰蛋白进行工程改造，同时保持适当的蛋白水平，因为工程化蛋白将从内源性启动子表达。在这里，我们提供了在细胞中对标记的、雷帕霉素可重定位的蛋白质进行工程改造的简单说明。该方案是在我们对微管解聚酶 MCAK/Kif2C 的工作背景下描述的，但它很容易适应其他基因和替代标签，如降解物、光遗传学构建体和其他实验有用的修饰。通过使用分裂 GFP 构建体测试最有效的靶位点，可以将脱靶效应降至最低。该方案不涉及专有试剂盒，只涉及可从库（如 addgene.org）获得的质粒。描述了使用内源蛋白水平进行验证、重定位和一些新发现的示例。有荧光显微镜的研究生应该能够使用该方案制备具有空间可控的内源蛋白的工程细胞。© 2023 作者。 Wiley Periodicals LLC 出版的《当代协议》。 基本方案 1：选择基因修饰的靶位点 基本方案 2：靶向基因修饰的 gRNA(s)的设计 基本方案 3：用于靶效率的分裂 GFP 测试 基本方案 4：重组模板和分析引物的设计 支持方案 1：用于分析 PCR 的引物设计 基本方案 5：工程细胞的转染、分离和验证 支持方案 2：与结合伴侣共转染工程细胞。
