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