Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA; Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA.
Department of Pharmacology, Vanderbilt University, Nashville, TN, USA; Vanderbilt Brain Institute, Vanderbilt University, Nashville, TN, USA.
STAR Protoc. 2024 Jun 21;5(2):103089. doi: 10.1016/j.xpro.2024.103089. Epub 2024 May 24.
Generating stable human embryonic stem cells (hESCs) with targeted genetic mutations allows for the interrogation of protein function in numerous cellular contexts while maintaining a relatively high degree of isogenicity. We describe a step-by-step protocol for generating knockout hESC lines with mutations in genes involved in synaptic transmission using CRISPR-Cas9. We describe steps for gRNA design, cloning, stem cell transfection, and clone isolation. We then detail procedures for gene knockout validation and differentiation of stem cells into functional induced neurons.
生成具有靶向基因突变的稳定人类胚胎干细胞 (hESC) 可以在保持相对高同源性的同时，在许多细胞环境中研究蛋白质功能。我们描述了使用 CRISPR-Cas9 生成涉及突触传递的基因突变的敲除 hESC 系的逐步方案。我们描述了 gRNA 设计、克隆、干细胞转染和克隆分离的步骤。然后，我们详细介绍了基因敲除验证和干细胞分化为功能性诱导神经元的程序。
