A Multiplexed Test for Bedaquiline-Resistant Tuberculosis using a Modified CRISPR-Cas13 System

Abstract

Tuberculosis (TB) remains the world’s single deadliest infectious disease in absolute terms, with drug-resistant strains posing a particularly difficult challenge for global health systems. Even bedaquiline (BDQ), the first novel anti-TB agent introduced in four decades and now the cornerstone of the WHO’s second-line treatment guidelines, has encountered alarming rates of emerging resistance. Thus, before subjecting a patient to the steep physical, financial, and time-intensive costs of BDQ-based regimens, it is imperative that drug susceptibility testing (DST) be performed to verify the efficacy of the drug against their specific strain. The only methods available, however, either require long incubation periods or rely on expensive genomic sequencing. Hence, there exists an urgent need for rapid and cost-effective BDQ DST. Here we present a novel molecular DST system that combines the precision of CRISPR-Cas13 with computational sequence design and microfluidic multiplexing to maximize accuracy, efficiency, and modularity. Through extensive screening, we assembled a panel of 60 guide RNAs that successfully distinguished between 81 of the most prevalent BDQ-resistant variants. Recognizing the need to further improve Cas13’s mismatch intolerance, both for DST and other RNA detection tasks, we also evaluated the viability of phosphorothioate (PS) crRNAs as a tool for specificity enhancement. After multiple rounds of rational design and systematic testing, we found that PS modifications offer a substantial discrimination boost across the entire crRNA-target duplex. However, significant disparities exist in the magnitude of the specificity improvement generated by different PS-modified sites and received at different mutation sites, potentially due to variation in Cas13 contact. Despite the need for further optimization, this proof of principle offers a promising path forward for robust single-nucleotide precision with Cas13.