AAAR 36th Annual Conference October 16 - October 20, 2017 Raleigh Convention Center Raleigh, North Carolina, USA
Abstract View
Laser-Induced Fluorescence Measurements of CRISPR-Cas9 Bioaerosols
BRIAN DAMIT, Elizabeth Corson, Mellisa Theodore, Ellen Forsyth, Rebecca Lilly, Robert Player, Robert M. Miceli, Johns Hopkins University Applied Physics Laboratory
Abstract Number: 767 Working Group: Bioaerosols
Abstract Advances in synthetic biology have the potential to transform the biodefense landscape. The advent of CRISPR-Cas genome editing tools can greatly enable development of designer biothreats by conferring more virulent properties, greater resistance to antibiotics, or enhanced transmission throughout a targeted population. Although studies have begun to explore the implications of CRISPR-Cas in the engineering of bioagents, negligible attention has been given to predicting the performance of fluorescent-based bioaerosol sensors against such emerging synthetic threats. The work here measured the laser-induced fluorescence (LIF) signatures of bacteria containing the CRISPR-Cas9 machinery in bioaerosols with time-of-flight LIF aerosol sensors, and compared the results to the signatures obtained from control microorganisms. CRISPR-Cas9-containing microbes were created by transformation of a GFP-expressing E. coli (ATCC 25922GFP) with plasmids containing genes for expression of 1) the Cas-9 enzyme (Addgene) and 2) sgRNA specific for the GFP target (Addgene). Test microbes were aerosolized with a Sono-Tek nozzle and sampled isokinetically by three LIF sensors: an Ultraviolet Aerodynamic Particle Sizer (UV-APS), a Wideband Integrated Bioaerosol Sensor (WIBS-4A), and a FLIR IBAC. Each sensor employed a different excitation wavelength and emission band thereby allowing comprehensive analysis of fluorescence discrepancies between test microbes. Microbes were aerosolized in both pure water and lysogeny broth. Generally, testing demonstrated that the addition of the plasmids negligibly altered fluorescence across all test sensors. For bioaerosol generation from lysogeny broth, fluorescence output was dominated by fluorescent materials contained in the broth rather than from microbes. Results from this study suggest that CRISPR/Cas9 plasmids and potentially CRISPR edits to the cell genome may not be discerned by LIF aerosol sensors.