Collaborative Research: Adapting Cas9 Protein from CRISPR as a Structural Unit for Molecular Assembly

第一部分： 即使使用非常先进的光刻技术，人类工程学也难以或不可能达到并控制低纳米长度尺度的物质。然而，生物系统利用分子相互识别和结合的能力来自组装非常复杂的结构，其特征尺寸在纳米范围内。从生物学中观察到的丰富多样的功能结构中获得灵感，并利用工程生物材料的新发现，该项目将扩大对生物材料及其可编程组装的基本理解。这项研究的成功完成将在短期内使使用蛋白质和核酸的纳米级自组装取得重大进展。从长远来看，这将为实现用于计算、通信和传感应用的越来越小的设备、植入式医疗设备和生物传感器以及可编程的人造分子机器提供所需的技术。该项目将为研究生和博士后学生提供尖端分子工程和生物纳米技术的培训机会。使用NSF？的补充资金机制，高中和本科生将被吸引到该项目参与研究，并在纳米科学的新兴领域的培训。该项目将扩大我们对纳米尺度现象和材料的科学理解，并提高我们在这种长度尺度上设计和工程新功能材料的能力。该项目的结果可能会影响电子设备的可持续制造以及医疗诊断和治疗新方法的未来应用领域。第二部分： 技术摘要DNA？高可靠性和可编程的分子识别能力导致了基于DNA的纳米技术领域，也被称为结构DNA纳米技术。这一领域的研究人员开发了DNA引导的分子和纳米尺度自组装的材料和技术，并取得了显着的最新进展，包括以前所未有的精度和平行度排列物质，蛋白质和金属颗粒的纳米尺度组织，以及人工分子机器的迷人演示。限制DNA纳米技术的一个问题？从原型演示到商业上可行的应用的翻译一直缺乏通用的、快速可重编程的方法来用多肽功能化DNA结构。来自CRISPR RNA指导的细菌免疫系统的Cas9蛋白在这里被用作解决这个问题的新方法。Cas9是一种可编程的蛋白质，其充当用于切割非自身核酸靶标的内切核酸酶。在工程化形式中，dCas9已被突变以结合DNA序列（由RNA分子指定）而不切割靶向DNA。dCas9蛋白、向导RNA及其靶DNA序列是很好理解的、模块化的和可编程的。因此，dCas9非常适合用于开发具有设计序列特异性的新分子组装工具家族，并且能够作为新的智能胶用于其他纳米材料的程序化组装，包括蛋白酶，亲和肽，无机纳米颗粒和碳纳米管。该项目的总体目标是将dCas9的可编程识别和结合功能添加到基于DNA的纳米技术可用的材料和方法的不断增长的工具箱中。具体而言，该项目旨在开发带有突变Cas9结构域的融合蛋白，以在生物医学应用以及电子和光子器件的生物纳米制造中以编程模式在DNA纳米结构上组织其他多肽（包括配体，体外选择的亲和肽，酶和抑制剂）。

PART 1: NON-TECHNICAL SUMMARY Even with very advanced lithographic techniques, it is difficult or impossible for human engineering to reach down and control matter in the low nanometer length-scale. However, biological systems utilize the ability of molecules to recognize and bind to one another for self-assembling very complex structures with feature sizes down in the nanometer range. Taking inspiration from the rich diversity of functional architectures observed in biology and making use of new discoveries in engineered biomaterials, this project will expand basic understanding of biomaterials and their programmable assembly. Successful completion of the research will, in the short-term, enable significant progress in nano-scale self-assembly using proteins and nucleic acids. In the long-term, this will provide the needed technology for implementation of ever-smaller devices for computing, communications, and sensing applications; implantable medical devices and biosensors; and programmable, artificial molecular machines. This project will provide training opportunities for postgraduate and postdoctoral students in cutting-edge molecular engineering and bionanotechnology. Using NSF?s supplemental funding mechanisms, high school and undergraduate students will be attracted to the project to participate research and to train in the emerging field of nanoscience. This project will expand our scientific understanding of nanometer-scale phenomena and materials as well as to improve our ability to design and engineer new functional materials on this length-scale. The results of this project could impact future application areas in the sustainable fabrication of electronic devices and new approaches to medical diagnostics and therapeutics. PART 2: TECHNICAL SUMMARY DNA?s capacity for highly reliable and programmable molecular recognition has led to the field of DNA-based nanotechnology, also known as structural DNA nanotechnology. Researchers in this field develop materials and techniques for DNA-guided molecular and nano-scale self-assembly and have made remarkable recent progress, including the ordering of matter with unprecedented precision and parallelism, nano-scale organization of proteins and metal particles, as well as fascinating demonstrations of artificial molecular machines. One problem that has limited DNA nanotech?s translation from prototype demonstrations to commercially viable applications has been the lack of a general purpose, rapidly-reprogrammable method for functionalizing DNA structures with polypeptides. The Cas9 protein from the CRISPR RNA-directed bacterial immune system is used here as a novel solution to this problem. Cas9 is a programmable protein that acts as an endonuclease for cleaving non-self nucleic acid targets. In an engineered form, dCas9 has been mutated to bind a DNA sequence (specified by an RNA molecule) without cleavage of the targeted DNA. The dCas9 protein, guide RNA, and its target DNA sequences are well understood, modular, and programmable. Consequently, dCas9 is perfect for use in the development of a new family of molecular assembly tools with designed sequence specificities and the ability to act as a new smart glue for the programmed assembly of other nanomaterials including protein enzymes, affinity peptides, inorganic nanoparticles, and carbon nanotubes. The overarching goal of the project is to add the programmable recognition and binding functions of dCas9 to the growing toolbox of materials and methods available to DNA-based nanotech. Specifically, the project seeks to develop fusion proteins bearing mutant Cas9 domains to organize other polypeptides (including ligands, in vitro selected affinity peptides, enzymes, and inhibitors) on DNA nanostructures in programmed patterns for biomedical applications as well as in the bionanofabrication of electronic and photonic devices.