Discipline: Biological Sciences
Nora J. Gilliam - Indiana University - Purdue University Indianapolis
Co-Author(s): Gordon C. Adams, Broad Institute of MIT and Harvard, MA; Jacob E. Lemieux, MD, DPhil, Massachusetts General Hospital, MA; Pardis C. Sabeti, MD, DPhil, Harvard University
CRISPR-based diagnostic tools have the potential to surpass the limits of current diagnostic tools with efficient, field-deployable technology and transform tick-borne infection clinical care and clinical diagnosis more broadly. Previous diagnostic tools have either been limited in sensitivity or required consistent electricity and sophisticated laboratory equipment, which are not always available in a resource-limited or field setting. SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) is a CRISPR-based diagnostic tool that couples isothermal recombinase polymerase amplification (RPA) of target genetic material and detection of the amplified material by CRISPR-Cas13a that can then be visualized on lateral-flow paper to achieve sensitivity and field-deployability. A remaining challenge with SHERLOCK is multiplex testing for multiple possible infections; however, RPA has been shown to amplify nonspecifically, which generates undesired products and introduces design and efficiency challenges for multiplexed amplification. The United States has seen an increase in the number of cases of tick-borne disease, but these infections cannot be distinguished on clinical grounds alone and patients are frequently infected by more than one tick-borne pathogen. Thus, there is a need for efficient and specific multiplex testing for detecting multiple tick-borne pathogens. In order to fulfill the need for specific and multiplexable isothermal amplification, other isothermal amplification methods should be explored and compared to RPA. We found that the isothermal amplification method helicase dependent amplification (HDA): 1) is multiplexable with three different tick-borne pathogens, Anaplasma phagocytophilum, Babesia microti, and Borrelia miyamotoi, 2) is compatible with CRISPR-Cas13a detection by allowing HDA products to be entered directly into the Cas13a detection step, 3) can visually amplify more specifically than RPA via gel electrophoresis, and 4) can amplify target genetic material from both synthetic genetic material of tick-borne pathogens and clinical samples from patients who have tested positive for babesiosis via traditional diagnostic measures. These findings demonstrate HDA?s potential to be used in SHERLOCK, but more optimization needs to be conducted in order to introduce an even more sensitive amplification step to SHERLOCK. Further experiments will elucidate the limits of sensitivity and detection for HDA when being multiplexed. The outcome of these experiments will lead to further optimization and validation for SHERLOCK technology as a novel approach to clinical detection of tick-borne pathogens. References: Gootenberg, J.S., Abudayyeh, O.O., et al. 2017. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 356(6336): 438-442. Myhrvold, C., Freije, C., Gootenberg, J.S., et al. 2018. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 360(6387): 444-448.
Funder Acknowledgement(s): This research was supported by the National Human Genome Research Institute by the NHGRI R25 DAP research grant through the Broad Institute Summer Research Program (BSRP). I thank the Sabeti Lab and the Broad Institute for granting me the opportunity to intern during the summer.
Faculty Advisor: Pardis Sabeti, email@example.com
Role: I conducted all of the PCR reactions, HDA reactions, and gel electrophoreses, and performed some of the CRISPR-Cas13a detection assays.