Amplification-free detection of specific Leishmania species using CRISPR-Cas12

About This Grant

PROJECT SUMMARY/ABSTRACT Leishmaniasis is a neglected tropical disease caused by over 20 protozoal species and transmitted by 70 species of sandflies, resulting in mucocutaneous, cutaneous, and visceral manifestations. Accurate species-level identification is essential for effective treatment and prognostication. Traditional microscopy lacks sensitivity and species specificity, whereas PCR, the most accurate current method, requires complex equipment and trained personnel in conventional laboratory settings. CRISPR-Cas direct detection technology, specifically CRISPR-Cas12, presents a promising alternative for species-specific diagnosis at the point of care. This technology detects DNA in vitro using a guide RNA that produces a fluorescent signal upon recognizing target DNA sequences, providing a faster and simpler alternative to PCR that could be used at the point of care. The objective of this research is to develop a CRISPR-based assay capable of speciating Leishmania, leveraging our expertise in CRISPR diagnostics for infectious diseases. This assay aims to enhance disease management by enabling accurate, rapid, and accessible species-level diagnosis. The project will achieve this by (1) identifying species-specific sequences for CRISPR-Cas targeting, and (2) developing and confirming Cas12 guides that can speciate Leishmania spp. DNA samples. These objectives will be accomplished through Cas12 guide design and selection, optimization of the CRISPR assay, and sample preparation to quantify parasite DNA spiked into blood. The anticipated outcomes include species-specific guides that be used for speciation, thereby informing treatment decisions. Additionally, the research will establish a pipeline for guide design applicable to further diagnostic research on Leishmaniasis and other neglected tropical diseases. The ultimate goal is to integrate this assay into a diagnostic platform suitable for low-resource settings, accelerating efforts to elucidate epidemiological patterns of leishmaniasis and guide treatment strategies aimed at disease elimination. These research objectives will be pursued in the context of a comprehensive research training program at the University of California, San Francisco, and Berkeley. This program encompasses structured technical and conceptual mentorship from the primary sponsor, Dr. Daniel Fletcher, an expert in diagnostics for neglected tropical diseases, and the co-sponsor, Dr. Dawn Wetzel, a physician-scientist expert in molecular parasitology. The applicant will receive additional training through structured preceptorships and regular advising sessions with physician-scientists specializing in the epidemiology and diagnostics of neglected tropical diseases, with a focus on pediatric care. This combination of experiences will provide invaluable training for an aspiring infectious disease physician-scientist.