Department of Anatomy and Embryology, Leiden University Medical Center, Leiden, The Netherlands.
Methods Mol Biol. 2022;2454:531-557. doi: 10.1007/7651_2021_368.
Advances in genome editing and our ability to derive and differentiate human induced pluripotent stem cells (hiPSCs) into a wide variety of cell types present in the body is revolutionizing how we model human diseases in vitro. Central to this has been the development of the CRISPR/Cas9 system as an inexpensive and highly efficient tool for introducing or correcting disease-associated mutations. However, the ease with which CRISPR/Cas9 enables genetic modification is a double-edged sword, with the challenge now being to introduce changes precisely to just one allele without disrupting the other.In this chapter, we describe strategies to introduce specific mutations into hiPSCs without enrichment steps. Monoallelic modification is contingent on the target activity of the guide RNA, delivery method of the CRISPR/Cas9 components and design of the oligonucleotide(s) transfected. As well as addressing these aspects, we detail high throughput culturing, freezing and screening methods to identify clonal hiPSCs with the desired nucleotide change. This set of protocols offers an efficient and ultimately time- and labor-saving approach for generating isogenic pairs of hiPSCs to detect subtle phenotypic differences caused by the disease variant.
基因组编辑技术的进步以及我们将人类诱导多能干细胞（hiPSCs）分化为体内多种细胞类型的能力，正在彻底改变我们在体外模拟人类疾病的方式。其中的核心是 CRISPR/Cas9 系统的发展，它是一种廉价且高效的工具，可用于引入或纠正与疾病相关的突变。然而，CRISPR/Cas9 实现基因修饰的便利性是一把双刃剑，现在的挑战是如何在不破坏其他基因的情况下，精确地将突变引入到仅一个等位基因中。在这一章中，我们描述了在不进行富集步骤的情况下将特定突变引入 hiPSCs 的策略。单等位基因修饰取决于向导 RNA 的靶向活性、CRISPR/Cas9 组件的递送方法和转染的寡核苷酸的设计。除了解决这些方面的问题，我们还详细介绍了高通量培养、冷冻和筛选方法，以鉴定具有所需核苷酸变化的克隆 hiPSCs。这组方案提供了一种高效且最终节省时间和劳动力的方法，可用于生成具有相同遗传背景的 hiPSC 对，以检测由疾病变异引起的微妙表型差异。
