Department of Genetics, Harvard Medical School, Boston, 02115 MA, USA, Biological and Biomedical Sciences Program, Harvard Medical School, Boston, 02115 MA, USA, Children's Hospital, Boston, 02115 MA, USA, Chemistry and Chemical Biology program, Harvard, 02138 Cambridge, MA, USA and Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, 02138 MA, USA.
Nucleic Acids Res. 2013 Oct;41(19):9049-61. doi: 10.1093/nar/gkt555. Epub 2013 Jul 31.
Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.
高效的人类诱导多能干细胞（hiPSC）基因组精确编辑策略将使复杂的基因组工程能够用于研究和临床目的。可编程序列特异性核酸酶如转录激活因子样效应物核酸酶（TALENs）和 Cas9-gRNA 的开发使得在感兴趣的靶向位点更有效地进行基因修饰成为可能。然而，仍有许多机会可以优化这些工具并扩大它们的应用范围。我们提出了一些改进方法：首先，我们开发了功能性重新编码的 TALEs（reTALEs），不仅能够实现简单的一锅式 TALE 合成，还可以使用慢病毒载体进行基于 TALE 的应用。然后，我们比较了 15 对靶向 CCR5 的 reTALENs 和 Cas9-gRNA 在 hiPSCs 中介导的基因组编辑效率，并结合两种方法优化了 ssODN 的设计，以引入特定的突变。我们发现，当与 ssODN 供体结合使用时，Cas9-gRNA 在 hiPSCs 中的非同源末端连接效率（3%）比 reTALENs（0.4%）高 7-8 倍，同源定向修复效率（1.0 比 0.6%）略高。使用最优设计，我们展示了一种在 3 周内生成无缝基因组校正 hiPSC 的简化流程。
