Laboratory of Bacteriology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA, Department of Microbiology and Immunobiology, Harvard Medical School, 4 Blackfan Circle, Boston, MA 02115, USA, Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA, McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA and Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Nucleic Acids Res. 2013 Aug;41(15):7429-37. doi: 10.1093/nar/gkt520. Epub 2013 Jun 12.
The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications.
人工控制转录的能力对于研究基因功能以及构建具有所需特性的合成基因网络都是至关重要的。Cas9 是一种 RNA 指导的双链 DNA 核酸酶，参与 CRISPR-Cas 对原核病毒的免疫防御。我们描述了 Cas9 核酸酶突变体的使用，该突变体保留了 DNA 结合活性，并可通过防止 RNA 聚合酶 (RNAP) 与启动子序列结合而被工程化为可编程转录抑制剂，或通过阻止正在运行的 RNAP 而成为转录终止子。此外，将 RNAP 的 ω 亚基与定向结合上游启动子区域的 Cas9 核酸酶突变体融合，可以实现可编程的转录激活。该技术实现的简单高效的基因表达调控，是研究基因网络以及开发合成生物学和生物技术应用的有用工具。
