CRISPRing Tuberculosis: Potential Application of a CRISPR-Based Technology in the Rapid Detection of Mycobacterium Tuberculosis
Access StatusFull text of the requested work is not available in DASH at this time ("restricted access"). For more information on restricted deposits, see our FAQ.
MetadataShow full item record
CitationKautu, Bwarenaba. 2020. CRISPRing Tuberculosis: Potential Application of a CRISPR-Based Technology in the Rapid Detection of Mycobacterium Tuberculosis. Master's thesis, Harvard Medical School.
AbstractTuberculosis (TB) is a deadly infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). Currently, it is the leading infectious disease killer in the world ranking above HIV/AIDS. Furthermore, the continued emergence and spread of drug-resistant forms of TB are increasingly a problem, causing increased mortality and global economic loss. One of the fundamental gaps that exists in the control of TB is the lack of rapid, ultra-sensitive, inexpensive, and easy-to-use diagnostic tools. The consequences of this setback are particularly acute in remote and low-income nations.
While there have been significant advances in the TB diagnostic development pipeline, control and management of TB remain a huge problem in many parts of the world. For instance, in remote and resource-limited settings, the lack of access to TB testing, coupled with high costs of tests, present significant barriers to TB elimination. Moreover, many existing TB diagnostic tests are known for their poor sensitivities, specificities, as well as lack of portability and ability to rapidly identify and discriminate among TB pathogens. Rapid and portable diagnostics that can both identify TB pathogens and provide drug susceptibility data in real-time would transform patient management and critical public health issues such as the current antibiotic resistance crisis. Their application would reach broadly from clinics and primary care offices to tertiary care hospitals, providing immediate guidance for therapeutic intervention thereby resulting in more prudent and appropriate use of antibiotics, in some cases with mortality benefit.
The objective of this thesis work is to explore the utility of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based technology in the rapid detection of TB and point-of-care applications. Here, we exploited the CRISPR-Cas-based RNA-detecting platform termed SHERLOCK (Specific High sensitivity Enzymatic Reporter unLOCKing) which utilizes a programmable RNAse (Cas13a) in the detection of genetic targets. We capitalize on targeting a multicopy mobile genetic element IS6110 to rapidly identify M. tuberculosis. Following in vitro transcription and deployment of Cas13a-CRISPR RNA complex, a robust, positive SHERLOCK signal was observed in the presence of fragmented and intact genomic DNA of the H37Rv strain. Ongoing experiments currently focus on developing multiplexing strategies to improve the Limit of Detection (LOD).
In the context of patient care, it is envisioned that the CRISPR-based test would be applicable in multiple clinical settings, including latent TB screening in communities with high TB burden, as well as triage testing of high-risk patients according to WHO’s recommendations. Characteristics of the triage TB test would include low cost, rapid turnaround time, easy-to-use, high sensitivity and specificity (comparable to standard confirmatory testing), good stability in a broad range of temperature and humidity, versatility in testing non-sputum samples, and usability in peripheral health facilities.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365251