Designing a Cas9/gRNA-assisted quantitative Real-Time PCR (CARP) assay for identification of point mutations leading to rifampicin resistance in the human pathogen Mycobacterium tuberculosis

A simple, rapid and low-cost diagnostic test, which can detect both the drug-sensitive and the drug-resistant tuberculosis (TB) cases is the need of the hour. Here, we developed a Cas9/gRNA-assisted quantitative Real-Time PCR (qRT-PCR) (CARP) assay to detect single nucleotide mutations causing drug resistance in the TB pathogen, Mycobacterium tuberculosis (Mtb). Guide RNAs (gRNAs) were designed against S531 and H526 positions in the rifampicin (RIF)-resistance-determining region (RRDR) of the Mtb rpoB gene that exhibit frequent mutations in the RR clinical isolates of Mtb. Conditions were optimised for in vitro Cas9 cleavage such that single nucleotide changes at these positions can be recognised by Cas9/gRNA complex with high sensitivity and 100% specificity. Further estimation of Cas9/gRNA-based cleavage of target DNA by qRT-PCR led to rapid detection of drug-resistant sequences. The newly designed CARP assay holds a great deal of promise in the diagnosis and prognosis of patients suffering from TB, in a cost-effective manner.