Harmful algal blooms (HABs) threaten drinking water, recreational waters, aquatic ecosystems, and local economies. Rapid molecular detection of cyanotoxin-producing cyanobacteria can support early warning and timely response, but most environmental CRISPR-Cas12a sensors still require nucleic acid preamplification to reach field-relevant sensitivity. This requirement increases assay time, operational complexity, and contamination risk. Autocatalytic CRISPR-Cas12a sensing offers a promising route to eliminate preamplification and achieve ultrarapid, ultrasensitive detection, but rapid positive feedback can cause early signal saturation and limit quantitative performance. This limitation is especially important for environmental monitoring, where contaminant concentrations often need to be compared with health- or management-relevant thresholds.
This project will develop a rapid, quantitative, preamplification-free CRISPR-Cas12a sensing platform for HAB monitoring by tuning autocatalytic reaction rates using caged crRNA scaffolds. Preliminary data show that the Dr-8 caged scaffold strongly suppresses Cas12a trans-cleavage activity while preserving a target concentration-dependent fluorescence response. The project will optimize caged crRNA scaffold designs and reaction time, validate the assay for two cyanotoxin-related genetic markers, mcyE and cyrJ, and convert the optimized assay into a lyophilized format for portable testing. The lyophilized assay will be demonstrated using HAB-impacted water samples through partnerships with PAHL, NYSFOLA, and SUNY ESF. Expected outcomes include an optimized quantitative autocatalytic CRISPR-Cas12a assay, a portable lyophilized sensing workflow for HAB screening, preliminary data for future NSF proposal development, and strengthened partnerships for community-engaged water monitoring.