Department of Genetics, Microbiology and Statistics, Faculty of Biology, University of Barcelona, CIBERER, IBUB, IRSJD, Barcelona, Spain.
Division of Neurology, Department of Clinical Sciences, Faculty of Medicine, Lund Stem Cell Center, Lund University, Lund, Sweden.
Methods Mol Biol. 2022;2549:409-425. doi: 10.1007/7651_2021_374.
The discovery that the CRISPR/Cas9 system could be used for genome editing purposes represented a major breakthrough in the field. This advancement has notably facilitated the introduction or correction of disease-specific mutations in healthy or disease stem cell lines respectively; therefore, easing disease modeling studies in combination with differentiation protocols. For many years, variability in the genetic background of different stem cell lines has been a major burden to specifically identify phenotypes arising uniquely from the presence of the mutation and not from differences in other genomic regions.Here, we provide a complete protocol to introduce random indels in human wild type pluripotent stem cells using CRISPR/Cas9 in order to generate clonal lines with potential pathogenic alterations in any gene of interest. In this protocol, we use transfection of a ribonucleoprotein complex to diminish the risk of off-target effects, and select clonal lines with promising indels to obtain disease induced pluripotent stem cell lines.
CRISPR/Cas9 系统可用于基因组编辑目的的发现是该领域的重大突破。这一进展显著促进了在健康或疾病干细胞系中分别引入或纠正特定疾病突变；因此，结合分化方案，减轻疾病建模研究的难度。多年来，不同干细胞系遗传背景的可变性一直是一个主要的负担，因为它难以专门确定独特地源于突变存在而不是源于其他基因组区域差异的表型。在这里，我们提供了一个完整的方案，使用 CRISPR/Cas9 在人类野生型多能干细胞中引入随机插入缺失，以产生具有任何感兴趣基因潜在致病性改变的克隆系。在本方案中，我们使用核糖核蛋白复合物的转染来降低脱靶效应的风险，并选择具有有希望的插入缺失的克隆系，以获得诱导性多能干细胞系。
