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

就在最近几年，Cas9已经成为分子生物学家工具箱中必不可少的工具。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，对其活性的精确控制以及其在复杂分子网络中的掺入一直是缺乏的。这项研究项目的目标是通过使用DNA纳米技术和微流控技术的工具来更好地控制Cas9。基于实验室在分子编程和微流控方面的专业知识，已经开发出基于RNA的新机制来控制酶的活性。这位研究人员首先采用了一种荧光分析方法来快速、定量地评估RNA指南的活性。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。在这项测试中，他发现，形成短发夹的特定区域的连接点对Cas9的活动是必不可少的。然后，他展示了在外源miRNA的控制下对指南的变构调节。最后，利用本发明，他设计了几个执行逻辑运算的转录网络，如异或、与或或门。

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.