CRISPR-integrated Biosensors for Human Health: Novel strategies for pathogen detection and cancer screening

Abstract

Rapid and accurate detection of pathogens and genetic mutations is essential for improving public health, guiding clinical decisions, and ensuring food safety. CRISPR-based biosensors offer a promising solution due to their programmability, sensitivity, and versatility. This dissertation focuses on the development of CRISPR-integrated biosensing platforms for assessing antimicrobial resistance, detecting bacterial pathogens, and identifying cancer-related genetic mutations. Five diagnostic systems were developed using different CRISPR/Cas technologies. First, a CRISPR/Cas9-engineered T4 bacteriophage was used for rapid antimicrobial susceptibility testing in Escherichia coli (E. coli). Second, a CRISPR/Cas12a-based electrochemical sensor was designed for the detection of Salmonella Typhimurium in food samples. Third, a CRISPR/Cas14a system combined with blocker displacement amplification enabled sensitive detection of the BRAF V600E mutation in colorectal cancer cells. Fourth, a one-pot CRISPR/Cas13 biosensor was designed for simultaneous detection of E. coli O157:H7 serotype markers, validated in a mouse infection model. Finally, the sensitivity of the CRISPR/Cas12a system was enhanced through site-specific covalent crosslinking between Cas12a and crRNA, which improved detection of SARS-CoV-2 in clinical samples. Together, these studies demonstrate the potential of CRISPR-based biosensors as powerful tools for diagnostics. The systems developed in this work are adaptable, sensitive, and suitable for applications in clinical diagnostics, food safety, and disease monitoring.