Photo Controlled CRISPR/Cas12a Microdroplets for Amplification-free Nucleic Acid Detection

Abstract Microdroplet platforms provide an amplification-free CRISPR diagnostic method by confining reactions to enhance local target concentration, but spontaneous Cas12a cleavage during droplet generation can generate false-positive signals that compromise quantitative accuracy. To address this, LUCID (Light-Up Cas12a In microDroplets), a photoactivatable CRISPR/Cas12a microdroplet platform was developed for amplification-free, digital nucleic acid detection. The strategy employs a photocloaking modification on crRNA that temporarily blocks Cas12a activity. Brief UV irradiation after droplet formation synchronously activates trans-cleavage across all droplets, eliminating premature signal generation. The platform detected HPV16 DNA down to 100 copies/μL and exhibited excellent specificity against non-target sequences. LUCID accurately discriminated genotyped HPV16/18 in multiple cervical cancer cell lines. By uniquely integrating optical controllability, amplification-free operation, and digital readout, LUCID effectively eliminates false positives and enables absolute quantification, offering a versatile and robust platform for diverse nucleic acid detection applications.