Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.
Crispr-mediated Gene Targeting of Human Induced Pluripotent Stem Cells.
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DOI：
10.1002/9780470151808.sc05a08s35
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发表时间：
2015-01-01
影响因子：
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通讯作者：
Church, George M
中科院分区：
文献类型：
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作者：
Byrne, Susan M;Church, George M
CRISPR/Cas9核酸酶系统可以在特定序列上产生双链DNA断裂，从而高效而准确地破坏、切除、突变、插入或替换基因。然而，与永生化肿瘤细胞系相比，人类胚胎干细胞或诱导多能干细胞(IPSCs)更难转化，对DNA损伤的抵抗力更差。在这里，我们描述了一种优化的人类IPSCs基因组工程方案，使用简单的瞬时转移载体和/或单链寡核苷酸。利用该方法，我们获得了大于60%的转染率，基因中断效率为1-25%，基因插入/替换效率为0.5-10%，而不需要任何进一步的选择或浓缩步骤。我们还描述了如何设计和评估最佳的sgRNA靶点和供体靶向载体；通过单细胞FACS分选克隆单个IPSC，并对成功编辑的细胞进行基因分型。
CRISPR/Cas9 nuclease systems can create double-stranded DNA breaks at specific sequences to efficiently and precisely disrupt, excise, mutate, insert, or replace genes. However, human embryonic stem or induced pluripotent stem cells (iPSCs) are more difficult to transfect and less resilient to DNA damage than immortalized tumor cell lines. Here, we describe an optimized protocol for genome engineering of human iPSCs using a simple transient transfection of plasmids and/or single-stranded oligonucleotides. With this protocol, we achieve transfection efficiencies greater than 60%, with gene disruption efficiencies from 1-25% and gene insertion/replacement efficiencies from 0.5-10% without any further selection or enrichment steps. We also describe how to design and assess optimal sgRNA target sites and donor targeting vectors; cloning individual iPSC by single cell FACS sorting, and genotyping successfully edited cells.