Figure 1.

Cas3-operated nucleic acid detection (CONAN)

(A) Schematic representation of the CONAN in vitro nucleic acid-detection platform. The E. coli CRISPR-Cas3 complex contains Cas3, Cas5, Cas6, Cas7, Cas8, and Cas11 proteins and CRISPR RNA (crRNA), FQ-ssDNA, fluorophore, and quencher-labeled single-stranded DNA probe.

(B) Collateral ssDNA cleavage activity measured by incubation of EcoCas3-EcoCascade/crRNA complex with a 60-bp dsDNA activator containing a target sequence flanked by a PAM and an FQ-labeled ssDNA probe in reaction buffer containing MgCl₂, CoCl₂, and ATP for 10 min at 37 °C. CRISPR-Cas3 mediated collateral ssDNA cleavage after targeting hEMX1-dsDNA in fragments with PAM (AAG, red), but not in fragments with non-PAM (CCA, blue), quantitatively represented by relative fluorescent units (RFU) per min; increasing rate of RFU/min (right). Means (n = 3), and standard deviations.

(C) CONAN assay on isothermal RPA amplicon products (blue) detected a single copy of the EMX1 activator fragments (1.7 a.m.); RFU at 10 min. Means (n = 3), and standard deviations.

(D) CONAN RPA also detected a single-copy mTyr activator (1.7 a.m.) when mixed with mouse genomic DNA. Means (n = 3), and standard deviations.