The knowledge of how biological molecules interact with each other is essential to understanding their roles in sustaining life. Molecular interactions are commonly measured using optical techniques and specialized optical labels on the molecules of interest. Recent advances in modern electronics and nanoscale materials potentially allow continuous monitoring of molecular interactions in a more natural state without optical labels and instruments. The goal of this CAREER project is to develop a single-molecule high-speed nanoelectronic platform to better understand the function of CRISPR (clustered regularly interspaced short palindromic repeats)-associated enzymes known as "molecular scissors". These enzymes allow gene editing and have revolutionized many basic and applied research areas. The knowledge gained will be beneficial for many applications in CRISPR engineering, pharmaceutical drug discovery, clinical diagnostics, and agricultural science. The project will promote early research involvement and mentorship opportunities to a new generation of engineers and scientists pursuing a career of interdisciplinary research intersecting modern biology, nanotechnology and engineering.
The investigator’s scientific career vision is to explore the utility of nano-electronic systems to develop transformative and customizable biosensing platforms for pharmaceutical, clinical, and environmental applications. As part of this vision, this project focuses on the integration of CRISPR (clustered regularly interspaced short palindromic repeats) with high bandwidth graphene field effect transistors (gFETs) designed for single-molecule sensing. The platform provides unique electronic signatures representing the molecular interactions that happen concurrently between the CRISPR and target DNA/RNA at different timescales. The CRISPR-Cas system is a family of RNA guided enzymes that is widely used for gene editing as it is capable of double-stranded DNA binding and cleavage, producing insertions and deletions (INDELs) at specific loci within the genome in vivo. The application areas of CRISPR technology are rapidly extending beyond genome editing, such as targeted gene regulation, in vivo imaging, epigenetic modulation as well as nucleic acid detection for diagnostic applications. The goal of this CAREER proposal is to provide a tool to evaluate the enzymatic activity of newly discovered or engineered CRISPR-Cas enzymes to better understand their biology and the impact of CRISPR-Cas mutagenesis, guide RNA modifications as well as genetic variation of the target on their functions. Successful development of the single molecule high bandwidth gFET for CRISPR analysis coupled with trained machine learning models, will provide a tool for detailed analysis of the CRISPR interactions with its target sequence in terms of binding, cleavage, and release of the target in real-time as well as identification of the variables most important for predicting CRISPR function. The information obtained can direct the design and optimization of CRISPR-Cas enzymes toward enhancing their efficiency and safety in wide range of applications. The applications of the single-molecule high bandwidth gFET platform can be expanded for understanding the molecular interactions of other enzymes beyond CRISPR.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
