Development of in vitro transcription networks using CRISPR/Cas9 and microfluidic technologies

在最近几年，Cas9已经成为分子生物学家工具箱中的重要工具。这个竞争激烈的领域拥有许多潜在的应用，从基础研究工具到基因治疗。然而，缺乏对其活性的精确控制及其在复杂分子网络中的掺入。该研究项目的目标是通过使用DNA纳米技术和微流体工具来更好地控制Cas9。基于实验室在分子编程和微流体方面的专业知识，已经开发了新的基于RNA的机制来控制酶的活性。该研究人员首先采用荧光测定法来快速定量评估RNA指导的活性。然后，他测试了近300种不同的指南变体，几乎改变了指南的任何功能区域。在这次测试中，他发现nexus，一个形成短发夹的特定区域，对Cas9的活性至关重要。然后，他证明了在外源性miRNA的控制下对指导的变构调节。最后，利用这项发明，他设计了几个执行逻辑运算的转录网络，如XOR，AND或OR门。

In just a few recent years, Cas9 has become an essential tool in the toolbox of molecular biologist. This highly-competitive field holds a lot of potential applications, from fundamental research tools to gene therapy. However, a precise control of its activity, and its incorporation in complex molecular networks have been lacking. The goal of this research project was to gain better control over Cas9 by using tools from DNA nanotechnology and microfluidic. Building on the expertise of the laboratory in molecular programming and microfluidics, new RNA-based mechanisms to control the activity of the enzyme has been developed. The research fellow first adapted a fluorescent assay to quickly and quantitatively assess the activity of a RNA guide. He then tested close to 300 different variants of guides, altering almost any functional region of the guide. During this testing, he found that the nexus, a particular region that forms a short hairpin, was essential to the activity of Cas9. He then demonstrated allosteric modulation of the guide under the control of an exogenous miRNA. Lastly, using this invention, he designed several transcription networks that perform logic operation, such as XOR, AND or OR gates.