Lebar Tina, Jerala Roman
Department of Synthetic Biology and Immunology, Kemijski inštitut/National Institute of Chemistry, Ljubljana, Slovenia.
Methods Mol Biol. 2018;1772:191-203. doi: 10.1007/978-1-4939-7795-6_10.
Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.
转录调控处于许多细胞过程的核心，是细胞对不同外部和内部信号作出反应的结果。对选定基因转录的控制使得以前所未有的方式塑造细胞反应成为可能。虽然来自细菌、酵母、植物或其他细胞的正交转录因子已被用于将新的细胞逻辑引入哺乳动物细胞，但可设计的模块化DNA结合结构域的发现，如转录激活样效应物（TALEs）和CRISPR系统，使得几乎可以靶向任何选定的DNA序列。DNA靶向结构域与转录效应结构域的融合或条件性结合能够对几乎任何内源性或异位基因进行可控调节。此外，设计的调控因子可以连接到遗传回路中以实现复杂反应，如不同类型的布尔函数和开关。在本章中，我们描述了在哺乳动物细胞中使用基于TALE和CRISPR的设计转录因子实现高效转录调控的方案。
