Unified hybrid RNA-regulated CRISPR diagnostic for concurrent Mycobacterium tuberculosis detection and rifampicin resistance profiling.

CRISPR-based nucleic acid detection is challenged by protospacer adjacent motif (PAM) dependency, amplicon consumption and single-tube multiplexing barriers, and poor discrimination of single-nucleotide variations (SNVs). These limitations are particularly acute in detecting Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance mutations for tuberculosis (TB) diagnostics. Herein, we devised TB-SCANER, a broad-spectrum, one-pot CRISPR-Cas12a diagnostic platform for MTB detection and RIF resistance profiling. The assay employs two unlocking single-stranded RNAs (uLsRNAs) transcribed from RPA products to unlock four cis-cleavage-locked CRISPR-Cas12a systems via distinct locking single-stranded RNAs (LsRNAs), establishing a two-to-four hybrid logic: one uLsRNA carries a single unlocking module for the M channel (targeting mpb64 gene), whereas the other contains three tandem modules engaging LsRNAs for subregions R1, R2, R3 within rpoB gene. With genomic DNA, detection limits are 50 fg/reaction (∼10 copies) for mpb64 and 500 fg/reaction (∼100 copies) for rpoB. The assay reliably identified clinically dominant rpoB mutations (Leu511Pro, Asp516Gly, His526Tyr, Ser531Leu) and low-prevalence variants (Ser522Met, His526Arg, Leu533Pro). For cultured isolates, TB-SCANER was 100% concordant with Sanger sequencing for RIF resistance mutation profiling; in clinical samples, it matched Xpert for MTB detection (100%), outperformed culture sensitivity (100% vs. 93.8%), and was fully concordant with Sanger sequencing for RIF resistance, eliminating Xpert's false-positive while identifying all resistant strains. Collectively, TB-SCANER is a rapid, accurate, low-cost, one-pot CRISPR diagnostic that overcomes multiplexing barriers, enabling concurrent MTB detection and RIF resistance profiling particularly in resource-limited settings, and features built-in adaptability to other pathogens.