Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.
Highly Efficient CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.
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DOI：
10.1002/cpsc.64
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发表时间：
2019-02-01
影响因子：
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通讯作者：
French, Deborah L
中科院分区：
文献类型：
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作者：
Maguire, Jean Ann;Cardenas-Diaz, Fabian L;French, Deborah L
人类PSCs为多种疾病的基础生物学和基于细胞的治疗提供了巨大的潜力。使用CRISPR-Cas9技术操纵这些细胞基因组的能力通过为设计或纠正与疾病相关的突变提供了一个有价值的工具，从而扩大了这一潜力。由于CRISPR-Cas9产生靶向双链断裂的效率很高，一个主要的挑战是在一个等位基因上引入精确的遗传修饰，而不在非靶标等位基因上形成插入。为了克服这一障碍，我们描述了两个寡核苷酸的使用，一个表达序列变化，另一个保持正常序列。此外，我们简化了转染和筛选方法，使该方案在细胞数量较少的情况下有效，并限制了劳动密集型克隆传代的数量。该方案提供了一种简化的、技术上简单的方法，用于生成在干细胞中模拟人类疾病的有价值的工具。2018年John Wiley&Sons，Inc.
Human PSCs offer tremendous potential for both basic biology and cell-based therapies for a wide variety of diseases. The ability to manipulate the genome of these cells using the CRISPR-Cas9 technology has expanded this potential by providing a valuable tool for engineering or correcting disease-associated mutations. Because of the high efficiency with which CRISPR-Cas9 creates targeted double-strand breaks, a major challenge has been the introduction of precise genetic modifications on one allele, without indel formation on the non-targeted allele. To overcome this obstacle, we describe the use of two oligonucleotides, one expressing the sequence change, with the other maintaining the normal sequence. In addition, we have streamlined both the transfection and screening methodology to make this protocol efficient with small numbers of cells and to limit the amount of labor-intensive clone passaging. This protocol provides a streamlined and technically simple approach for generating valuable tools to model human disease in stem cells. © 2018 by John Wiley & Sons, Inc.