Li Shenglan, Xue Haipeng, Wu Jianbo, Rao Mahendra S, Kim Dong H, Deng Wenbin, Liu Ying
1 Department of Neurosurgery, Medical School, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.
2 Center for Stem Cell and Regenerative Medicine, the Brown Foundation Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center at Houston , Houston, Texas.
Stem Cells Dev. 2015 Dec 15;24(24):2925-42. doi: 10.1089/scd.2015.0131. Epub 2015 Nov 5.
Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼ 33% correctly targeted clones) compared to conventional targeting protocol (∼ 3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.
人诱导多能干细胞（hiPSC）技术是用于发育和疾病建模、药物筛选及再生医学的强大工具。在hiPSC中进行精确的基因靶向极大地促进了这些应用。我们开发了一种基于快速且精确的成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）技术的方法，并在hiPSC系中获得了针对重要的神经前体细胞转录因子神经生成素2（NEUROG2）的荧光蛋白和抗生素抗性双敲入报告基因。与传统靶向方案（约3%）相比，在同一基因座处，CRISPR/Cas9介导的同源定向重组中的基因靶向效率得到了极大提高（约33%正确靶向的克隆）。未检测到脱靶事件。此外，利用CRISPR/Cas9系统的多种应用，我们设计了反式激活组件以瞬时诱导NEUROG2表达，这有助于鉴定人类NEUROG2的转录因子结合位点和反式调节区域。在敲入谱系hiPSC报告平台中使用CRISPR/Cas9基因组编辑结合神经祖细胞的荧光激活细胞分选的策略可能广泛适用于其他干细胞衍生物和亚群。
