Murdoch Children's Research Institute, The Royal Children's Hospital, Parkville, Victoria, Australia.
Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria, Australia.
Nat Protoc. 2018 May;13(5):875-898. doi: 10.1038/nprot.2018.007. Epub 2018 Apr 5.
The utility of human induced pluripotent stem cells (iPSCs) is enhanced by an ability to precisely modify a chosen locus with minimal impact on the remaining genome. However, the derivation of gene-edited iPSCs typically involves multiple steps requiring lengthy culture periods and several clonal events. Here, we describe a one-step protocol for reliable generation of clonally derived gene-edited iPSC lines from human fibroblasts in the absence of drug selection or FACS enrichment. Using enhanced episomal-based reprogramming and CRISPR/Cas9 systems, gene-edited and passage-matched unmodified iPSC lines are obtained following a single electroporation of human fibroblasts. To minimize unwanted mutations within the target locus, we use a Cas9 variant that is associated with decreased nonhomologous end-joining (NHEJ) activity. This protocol outlines in detail how this streamlined approach can be used for both monoallelic and biallelic introduction of specific base changes or transgene cassettes in a manner that is efficient, rapid (∼6-8 weeks), and cost-effective.
人类诱导多能干细胞（iPSCs）的实用性通过一种能力得到增强，即能够精确修饰选定的基因座，而对剩余基因组的影响最小。然而，基因编辑 iPSC 的产生通常需要多个步骤，需要长时间的培养期和几个克隆事件。在这里，我们描述了一种从人成纤维细胞中一步生成克隆衍生的基因编辑 iPSC 系的可靠方法，无需药物选择或 FACS 富集。使用增强的基于附加体的重编程和 CRISPR/Cas9 系统，在对人成纤维细胞进行单次电穿孔后，可获得基因编辑和传代匹配的未修饰 iPSC 系。为了最大限度地减少靶基因座内的意外突变，我们使用与非同源末端连接（NHEJ）活性降低相关的 Cas9 变体。本方案详细介绍了如何以高效、快速（约 6-8 周）且具有成本效益的方式，使用这种简化方法对特定碱基变化或转基因盒进行单等位基因和双等位基因引入。
