CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.
CRISPR-Cas9-Based Genome Editing of Human Induced Pluripotent Stem Cells.
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DOI：
10.1002/cpsc.46
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发表时间：
2018-02-28
影响因子：
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通讯作者：
Tucker, Budd A
中科院分区：
文献类型：
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作者：
Giacalone, Joseph C;Sharma, Tasneem P;Tucker, Budd A
人诱导多能干细胞（hiPSCs）是自体细胞替代的理想细胞来源。然而，对于孟德尔疾病患者，在细胞分化和移植之前，可能需要对原始致病突变进行基因校正。CRISPR-Cas9系统的出现彻底改变了基因组编辑领域。通过引入设计和验证相对简单的廉价试剂，现在有可能纠正遗传变异或在基因组内的任何位置插入所需的序列。基于crispr的患者特异性iPSCs基因组编辑显示出未来自体细胞替代疗法的巨大前景。然而，需要注意的是，hipsc是出了名的难以转染的，优化的实验设计考虑通常是必要的。本单元描述了高效的基于crispr的患者特异性iPSCs基因组编辑的设计策略和方法。此外，它还详细介绍了一种灵活的方法，利用正选择生成具有所需基因组修饰的克隆，Cre-lox重组去除集成选择盒，负选择去除完整选择盒中残留的hipsc。©2018 by John Wiley & Sons, Inc。
Human induced pluripotent stem cells (hiPSCs) are the ideal cell source for autologous cell replacement. However, for patients with Mendelian diseases, genetic correction of the original disease-causing mutation is likely required prior to cellular differentiation and transplantation. The emergence of the CRISPR-Cas9 system has revolutionized the field of genome editing. By introducing inexpensive reagents that are relatively straightforward to design and validate, it is now possible to correct genetic variants or insert desired sequences at any location within the genome. CRISPR-based genome editing of patient-specific iPSCs shows great promise for future autologous cell replacement therapies. One caveat, however, is that hiPSCs are notoriously difficult to transfect, and optimized experimental design considerations are often necessary. This unit describes design strategies and methods for efficient CRISPR-based genome editing of patient- specific iPSCs. Additionally, it details a flexible approach that utilizes positive selection to generate clones with a desired genomic modification, Cre-lox recombination to remove the integrated selection cassette, and negative selectionto eliminate residual hiPSCs with intact selection cassettes. © 2018 by John Wiley & Sons, Inc.