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

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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.