CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells.
CRISPR/Cas9-Mediated Introduction of Specific Heterozygous Mutations in Human Induced Pluripotent Stem Cells.
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DOI：
10.1007/7651_2021_368
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Davis RP
中科院分区：
文献类型：
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作者：
Brandão KO;Grandela C;Yiangou L;Mummery CL;Davis RP
基因组编辑的进展以及我们将人类诱导多能干细胞（hiPSC）衍生和分化为体内多种细胞类型的能力正在彻底改变我们体外模拟人类疾病的方式。其中的核心是CRISPR/Cas9系统的开发，作为一种引入或纠正疾病相关突变的廉价且高效的工具。然而，CRISPR/Cas9使基因修饰变得容易是一把双刃剑，现在的挑战是在不破坏另一个等位基因的情况下精确地将变化引入一个等位基因。在本章中，我们描述了在没有富集步骤的情况下将特定突变引入hiPSC的策略。单等位基因修饰取决于指导RNA的靶活性、CRISPR/Cas9组分的递送方法和转染的寡核苷酸的设计。除了解决这些方面，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hiPSC。这组方案提供了一种有效且最终省时省力的方法，用于产生hiPSC的同基因对，以检测由疾病变体引起的细微表型差异。
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.