ERI: Enhancing CRISPR-Cas12a-Powered Biosensing Detection of the Microcystin Synthetase Gene for Early Warning of Harmful Algal Blooms Using Magnetic Bead-Based Reporter

Clarkson University, Potsdam NY

Investigators

Abstract

The frequent and pervasive occurrences of harmful algal blooms (HABs) in the United States and worldwide, which adversely impact ecosystem/human health and inflict economic damage, require innovative early detection methods to minimize and mitigate their impact. Traditional monitoring methods only identify toxins after a HAB event has occurred, thus reacting to rather than preventing the outbreak. Recognizing the urgent need for proactive environmental monitoring strategies, this ERI project will address this critical gap and introduce a transformative approach utilizing a novel biosensing technology, clustered regularly interspaced short palindromic repeats (CRISPR). The overarching goal of this project is to develop a highly sensitive and selective biosensor based on CRISPR-Cas12a technology coupled with a novel magnetic bead-based reporter for the early detection of the microcystin synthetase gene (mcy), the prerequisite in the production of the most prevalent cyanotoxins (i.e., microcystins) during HABs. The successful completion of this project will benefit society by advancing our capability to predict cyanobacterial HABs in a timely and accurate manner with the goal of reducing their adverse impacts on public health, ecosystems, and the economy. Additional benefits to society will be achieved through educational outreach activities and student training, including the mentoring of one graduate student and two undergraduate students at Clarkson University. Microcystins produced by toxic cyanobacteria during Harmful Algal Blooms (HABs) pose severe threats to human health and ecosystems and lead to enormous economic loss. Current monitoring methods focused on microcystins do not have proactive features as the toxins become detectable usually after the outbreak of a HAB event. A breakthrough in the monitoring methodology is identifying the microcystin synthetase (mcy) gene, the prerequisite of microcystin production by microcystin-producing strains. However, the mcy gene detection by conventional PCR-based methods requires significant capital investment, high operational costs, experienced personnel, and a long lead time. The goal of this ERI project is to develop a highly selective and sensitive biosensor for rapid and simplified detection of the mcy gene by implementing the revolutionary clustered regularly interspaced short palindromic repeats (CRISPR) technique with a novel magnetic bead (MB)-enhanced reporter. The specific objectives of the research are to 1) design and optimize the CRISPR-Cas12a/crRNA complex for the mcyE gene detection; 2) develop an MB-based reporter for enhanced CRISPR-Cas12a-derived fluorescence signals; 3) investigate the matrix effects and demonstrate the MB-enhanced CRISPR-Cas12a-powered biosensing system using lake water samples. To implement the education and outreach activities of the project, the Principal Investigator plans to leverage existing programs at Clarkson University and partnerships with the local children's museum and senior learning center to promote undergraduate research, K-12 STEM education, and life-long learning in the community. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.