Graduate Assistant II University of Maryland-College Park Pembroke, Massachusetts
Body of Abstract: CRISPR technology has revolutionized genome editing and transcriptional regulation in living organisms. Beyond these capabilities, CRISPR has also been developed to be a valuable tool in point-of-care pathogen detection. CRISPR-based detection strategies have the capacity to produce rapid, robust, and sensitive results on-site and without the usage of expensive technology or trained personnel. Utilizing the trans-cleavage activity of CRISPR-Cas12a nuclease upon cleaving its target sequence, researchers have developed CRISPR-based detection assays to identify the presence of particular sequences of target pathogens. In our lab, we have developed a CRISPR-Cas12a-based detection strategy for the emerging and widespread plant pathogen phytoplasma (Candidatus phytoplasma). Many conventional CRISPR-based detection methods require the pre-amplification of a portion of the pathogen genome to increase sensitivity limits, but this often leads to off-target amplification and false positives. Our developed assay is a pragmatic improvement upon these established methods which improved the sensitivity limit without the use of a pre-amplification step. Our system incorporates engineered Cas12a variants, sophisticated redesign of reporter oligonucleotides, and careful genomic consideration from over 7,000 phytoplasma sequences in the NCBI genomic database to select the most optimal target sites. We demonstrate that our CRISPR-based detection strategy is as sensitive and specific as current detection methods, like qPCR, with the potential to further push the detection limit. Based on these developed principles, our system has the potential to be used to detect any DNA sequence from the pathogen of interest, thus preventing the spread of plant disease.