Photocontrolled Programmable Enzymatic Cascade for Robust CRISPR Diagnostics

CRISPR-Cas12a-based diagnostic technologies have revolutionized nucleic acid detection, but their broader application remains constrained by the protospacer adjacent motif (PAM) requirement and limited multiplexing capabilities due to reliance on trans-cleavage. Here, we present a photocontrolled programmable enzymatic cascade strategy that enables temporal regulation of three sequential reactions─nucleic acid amplification, photoactivated lambda exonuclease (λ-exon)-mediated single-stranded DNA (ssDNA) generation, and PAM-independent Cas12a detection─all within a one-pot system, effectively overcoming the PAM constraint. We further exploit the orthogonal trans-cleavage activity of Cas12a and Cas13a to enable simultaneous dual-gene detection within the one-pot system, thereby circumventing multiplexing limitations. Applied to clinical Mycobacterium tuberculosis (MTB) samples, the method allows detection of both the IS6110 gene of MTB and the human ACTB (β-actin) internal control gene. This photocontrolled one-pot CRISPR diagnostic technology enhances flexibility in target site selection and overcomes the limitations of conventional CRISPR diagnostics, which cannot simultaneously detect both target genes and internal controls. This approach holds promise for advancing the clinical application of CRISPR-based diagnostics.