Feng Yuanxi, Zhang Siliang, Huang Xin
1] Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA [2] Department of Internal Medical Oncology, The Third Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150081, China [3].
1] Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA [2] Department of Radiation Oncology, The Third Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150081, China [3].
Sci Rep. 2014 Jan 10;4:3632. doi: 10.1038/srep03632.
Recently, transcription activator-like effector nucleases (TALENs) have emerged as a highly effective tool for genomic editing. A pair of TALENs binds to two DNA recognition sites separated by a spacer sequence, and the dimerized FokI nucleases at the C terminal then cleave DNA in the spacer. Because of its modular design and capacity to precisely target almost any desired genomic locus, TALEN is a technology that can revolutionize the entire biomedical research field. Currently, for genomic editing in cultured cells, two plasmids encoding a pair of TALENs are co-transfected, followed by limited dilution to isolate cell colonies with the intended genomic manipulation. However, uncertain transfection efficiency becomes a bottleneck, especially in hard-to-transfect cells, reducing the overall efficiency of genome editing. We have developed a robust TALENs system in which each TALEN plasmid also encodes a fluorescence protein. Thus, cells transfected with both TALEN plasmids, a prerequisite for genomic editing, can be isolated by fluorescence-activated cell sorting. Our improved TALENs system can be applied to all cultured cells to achieve highly efficient genomic editing. Furthermore, an optimized procedure for genomic editing using TALENs is also presented. We expect our system to be widely adopted by the scientific community.
最近，转录激活样效应因子核酸酶（TALENs）已成为一种用于基因组编辑的高效工具。一对TALENs与由间隔序列隔开的两个DNA识别位点结合，然后C末端的二聚化FokI核酸酶在间隔区切割DNA。由于其模块化设计以及精确靶向几乎任何所需基因组位点的能力，TALEN是一项可以彻底改变整个生物医学研究领域的技术。目前，对于培养细胞中的基因组编辑，共转染两个编码一对TALENs的质粒，随后进行有限稀释以分离具有预期基因组操作的细胞集落。然而，不确定的转染效率成为一个瓶颈，尤其是在难以转染的细胞中，降低了基因组编辑的整体效率。我们开发了一种强大的TALENs系统，其中每个TALEN质粒还编码一种荧光蛋白。因此，可以通过荧光激活细胞分选分离同时转染了两个TALEN质粒的细胞，这是基因组编辑的一个先决条件。我们改进的TALENs系统可应用于所有培养细胞，以实现高效的基因组编辑。此外，还介绍了一种使用TALENs进行基因组编辑的优化程序。我们期望我们的系统能被科学界广泛采用。
