CAREER: High bandwidth nano-transistors to understand the kinetic basis for CRISPR/CAS enzymes to enhance their applications for diagnostics and therapeutics

生物分子如何相互作用的知识对于理解它们在维持生命中的作用是至关重要的。通常使用光学技术和感兴趣的分子上的特殊光学标记来测量分子间的相互作用。现代电子学和纳米材料的最新进展可能使人们能够在更自然的状态下连续监测分子相互作用，而不需要光学标记和仪器。这个职业项目的目标是开发一个单分子高速纳米电子平台，以更好地了解CRISPR(簇状规则间隔短回文重复序列)相关酶的功能，这种酶被称为“分子剪刀”。这些酶允许基因编辑，并使许多基础和应用研究领域发生了革命性的变化。所获得的知识将有益于CRISPR工程、药物开发、临床诊断和农业科学的许多应用。该项目将促进新一代工程师和科学家的早期研究参与和指导机会，他们将从事跨越现代生物学、纳米技术和工程学的跨学科研究。研究人员的科学职业愿景是探索纳米电子系统的用途，以开发用于制药、临床和环境应用的变革性和可定制的生物传感平台。作为这一愿景的一部分，该项目专注于CRISPR(集群式规则间隔短回文重复序列)与专为单分子传感设计的高带宽石墨烯场效应管(GFET)的集成。该平台提供了独特的电子签名，代表了CRISPR和目标DNA/RNA之间在不同时间尺度上同时发生的分子相互作用。CRISPR-Cas系统是一个RNA引导的酶家族，广泛用于基因编辑，因为它能够与双链DNA结合和切割，在体内基因组中的特定位置产生插入和缺失(Indels)。CRISPR技术的应用领域正在迅速扩展到基因组编辑之外，如靶向基因调控、活体成像、表观遗传调节以及用于诊断应用的核酸检测。这项职业计划的目标是提供一种工具来评估新发现或改造的CRISPR-Cas酶的酶活性，以更好地了解它们的生物学和CRISPR-Cas突变的影响，指导RNA修饰以及靶标的遗传变异对其功能的影响。用于CRISPR分析的单分子高带宽GFET的成功开发与训练有素的机器学习模型相结合，将提供一种工具，用于详细分析CRISPR与其目标序列的结合、切割和实时释放，以及识别对预测CRISPR功能最重要的变量。所获得的信息可以指导CRISPR-CAS酶的设计和优化，以提高其在广泛应用中的效率和安全性。单分子高带宽GFET平台的应用可以扩展，以了解CRISPR以外的其他酶的分子相互作用。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.