Collaborative Research: ENG-BIOTECH: Mechanism-Guided Engineering of Thermophilic CRISPR-Cas13 for Ultrasensitive and Robust RNA Detection

Infectious diseases are a major global health concern. Rapid and accurate detection of the presence of disease-causing RNA is crucial for effective diagnosis and control of these diseases. The CRISPR-Cas13 system is a cutting-edge technology for RNA detection. However, the currently used Cas13 enzymes can become unstable and lose their effectiveness during long-term storage and in field applications. This project aims to improve the stability and sensitivity of a heat-resistant version of the Cas13 enzyme, making it more reliable and sensitive for detecting RNA. This includes enhancing the enzyme's ability to recognize and cut RNA and combine it with advanced electrochemical devices to create a highly sensitive and stable detection method. The proposed scientific advancements are closely connected to educational outreach activities. The project will involve high school and community college students, particularly from underrepresented backgrounds, in biological and bioengineering research. Students will receive training in experimental techniques, data analysis, and scientific writing. Additionally, high school students will be introduced to CRISPR technology through a biotech academy and integrate the research findings into university courses. The goal of the project is to combine mechanism-based protein engineering and cutting edge electrochemical devices to generate next-generation RNA detection tools for infectious disease diagnosis. The project will leverage the CRISPR-Cas13 system, which has shown great promise as next-generation diagnostics for in vitro RNA detection owing to its high specificity, programmability, and fast reaction rate. The collaborative project aims to first investigate the structure and mechanism of the recently discovered thermostable Cas13. Leveraging the mechanistic understanding, rational engineering of the thermostable Cas13 will be performed to produce new variants with superior thermostability and protease resistance, as well as enhanced target sensitivity and reaction speed. The engineered Cas13 variants will be combined with innovative electrochemical devices to enable ultrasensitive and robust RNA detection of various pathogens derived from clinical samples. Successful completion will provide superior RNA detection tools for medical and research applications, alongside novel insights into the Cas13 nuclease mechanism. 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.