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Modular CRISPR-diagnostics for infectious diseases
Infectious diseases continue to represent a significant fraction of worldwide disease burden. A critical part of lowering this burden entails effective testing strategies, as underscored by the ongoing COVID-19 pandemic. The goals of diagnostics, screening, and surveillance have demanded distinct yet innovative approaches to testing. In this work, I present two novel approaches for multiplexed nucleic acid detection (CARMEN and WATSON) that build on the existing CRISPR-diagnostic method SHERLOCK. SHERLOCK combines traditional amplification (PCR or isothermal) with target-specific CRISPR-Cas13 detection and enables new, modular assay designs. With CARMEN, we employ a droplet microfluidic platform to perform thousands of parallel SHERLOCK reactions in nanoliter droplets. CARMEN’s potential for high-throughput infectious disease screening is demonstrated through the design of detection assays for large panels of clinically relevant viral and bacterial pathogens, as well as resistance markers. With WATSON, we maximize the sensitivity of SHERLOCK by targeting multiple tiled regions across a single pathogen genome. The clinical significance of WATSON is demonstrated by applying it to the detection of plasma circulating cell-free DNA in tuberculosis, highlighting its potential as a liquid biopsy test for infectious disease management.
Thesis Supervisors:
Deborah T. Hung, MD, PhD
Professor of Genetics, HMS; Co-Director of the Infectious Disease and Microbiome Program, Broad Institute
Paul C. Blainey, PhD
Associate Professor of Biological Engineering, MIT; Core Institute Member, Broad Institute
Thesis Committee Chair:
Lee Gehrke, PhD
Hermann von Helmholtz Professor of Health Sciences and Technology, MIT; Professor of Microbiology and Immunobiology, HMS
Thesis Reader:
Yonatan Grad, MD, PhD
Associate Professor of Immunology and Infectious Diseases, HSPH
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