The COVID-19 pandemic has brought to light the importance of diagnostics in detecting and containing infectious diseases. Recently, a new class of CRISPR-Cas enzymes, known as Cas12, has been discovered and utilized as a biosensor to detect nearly any genetic biomarker of interest. Cas12 is similar to other CRISPR-Cas proteins, in that it forms an RNA-protein complex with crRNA, and the complex recognizes and cleaves target DNA determined by the crRNA sequence. Cas12 is unique in that upon recognition of target DNA, it becomes activated and non-specifically cleaves any nearby DNA. This non-specific cleavage property can be leveraged to create a biosensor by adding a reporter DNA substrate to the reaction. The DNA substrate is composed of a fluorophore-quencher pair, such that fluorescence is quenched and cannot be detected until the DNA substrate is cleaved. Upon cleavage, the fluorophore and quencher separate, resulting in an increase in fluorescence. In our system, Cas12 is programmed to recognize a specific sequence of the SARS-CoV-2 genome. This class of biosensor is useful because it can detect nucleic acids, yet provide a faster response compared to traditional PCR-based assays, which take days. Unfortunately, CRISPR-Cas biosensors still suffer from off-target activity, meaning that similar sequences can also activate the biosensor, thus resulting in false positive signal. The goal of this project is to systematically study factors that affect Cas12 activity. Specifically, we investigated the role of crRNA concentration, crRNA length, and mutation position and how they affect Cas12 activity. Ultimately, findings from this research will aid in developing next-generation biosensors to enable rapid point-of-care diagnostics for detecting and containing disease outbreaks. This research has been relevant to my future career as I would like to work in a forensic laboratory. This research has aided towards this goal as it encompasses several disciplines, including analytical chemistry, biochemistry, and molecular biology.
Author: Alexandra Bender, Chemistry Major
Advisor: Kevin Yehl, Chemistry and Biochemistry department
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