CRISPRD workflow for
multiplex detection of hrHPVs. (a) CRISPRD
is designed to detect three targets (HPV16, HPV16, and an internal
control) simultaneously. The two layers of amplification, LAMP for
DNA and T7-based transcription for RNA, are followed by CRISPR-based
specific detection of the three targets in one pot. CRISPRD deploys
three orthogonal Cas nucleases (TccCas13a, HheCas13a, and AapCas12b)
to detect their cognate target via gRNAs and digest its associated
fluorophore-conjugated DNA or RNA reporter only. Target-induced Cas
variant-based digestion of the reporter releases the fluorophore as
a visual fluorescent readout for pathogen detection. (b) From sample
to answer: after processing the sample, nucleic acid is loaded to
the one-pot CRISPRD reaction where amplification and detection occur
in a single step.