Menzies Institute for Medical Research, University of Tasmania, Tasmania, Australia.
Wicking Dementia Research and Education Centre, University of Tasmania, Tasmania, Australia.
Methods. 2021 Oct;194:18-29. doi: 10.1016/j.ymeth.2021.02.009. Epub 2021 Feb 17.
Induced pluripotent stem cells (iPSCs) have become widely used for disease modelling, particularly with regard to predisposing genetic risk factors and causal gene variants. Alongside this, technologies such as the CRISPR/Cas system have been adapted to enable programmable gene editing in human cells. When combined, CRISPR/Cas gene editing of donor-specific iPSC to generate isogenic cell lines that differ only at specific gene variants provides a powerful model with which to investigate genetic variants associated with diseases affecting many organs, including the brain and eye. Here we describe our optimized protocol for using CRISPR/Cas ribonucleoproteins to edit disease causing gene variants in human iPSCs. We discuss design of crRNAs and homology-directed repair templates, assembly of CRISPR/Cas ribonucleoproteins, optimization of delivery via nucleofection, and strategies for single cell cloning, efficient clone cryopreservation and genotyping for identifying iPSC clones for further characterization.
诱导多能干细胞（iPSCs）已广泛用于疾病建模，尤其是与易感性遗传风险因素和因果基因变异有关的疾病建模。除此之外，CRISPR/Cas 系统等技术已被改编用于实现人类细胞中的可编程基因编辑。当将供体特异性 iPSC 的 CRISPR/Cas 基因编辑与仅在特定基因变异处不同的同基因细胞系生成相结合时，提供了一种强大的模型，可用于研究与影响包括大脑和眼睛在内的许多器官的疾病相关的遗传变异。在这里，我们描述了我们优化的使用 CRISPR/Cas 核糖核蛋白编辑人类 iPSC 中致病基因变异的方案。我们讨论了 crRNA 和同源定向修复模板的设计、CRISPR/Cas 核糖核蛋白的组装、通过核转染进行优化的递送以及单细胞克隆、高效克隆冷冻保存和基因分型的策略，以鉴定用于进一步表征的 iPSC 克隆。
