Multiplexed detection of tuberculosis using CRISPR-Cas

Abstract

Tuberculosis (TB) is the world’s deadliest infectious disease and is caused by Mycobacterium tuberculosis (Mtb). TB is a persistent public health crisis, despite being both preventable and curable. Existing approaches to TB diagnostics are costly, time-intensive and difficult to deploy at scale. Co-occurring infectious disease outbreaks (such as the COVID-19 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SC2)) and drug-resistant strains further complicate the ability to provide TB diagnosis and care. Thus, comprehensive, accessible and multiplexed TB diagnostic platforms are needed to strengthen prevention and control efforts. Recent advancements in CRISPR-based diagnostics have led to significant breakthroughs in disease detection. Technologies such as Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN) and occluders have revolutionized the precision and scalability of pathogen identification and mutation detection (i.e., identifying mutations associated with the pathogen). Here, to address the burden of co-occurring infectious disease outbreaks, we present SHINE-TRI (SHINE-TRIplex), the first SHINE assay that can simultaneously detect three targets: Mtb, SC2 and human DNA. Additionally, to address the burden of drug-resistant Mtb, we present mCARMEN-RIF (mCARMEN-RIFampicin), which is capable of detecting the 24 mutations that are most predictive of rifampicin-resistant TB (RR-TB) using only six CRISPR RNAs (crRNAs) by leveraging a mutation clustering approach and improved occluder technology. Each of these two novel assays have the potential to contribute to the improvement of TB surveillance by enabling more informed decisions regarding patient care.