Fig. 4: Cas12j3 PAM recognition, uncoupling of the Watson–Crick dA-1:dT+1 pair and unzipping.

a Surface representation of Cass12j3–R-loop complex. The white dashed arrow shows the predicted path of the NT-strand to the nuclease site after dG-2 (Supplementary Fig. 6). b Detailed view of the PAM nucleotides recognition and dsDNA unwinding depicting the conserved K26, K30, Q123, and Q197 residues (Supplementary Fig. 5c). c Zoom of the dT+1/dA-1 pair uncoupling, phosphate inversion and unzipping (Supplementary Fig. 5d). d dsDNA cleavage assays using Cas12j3 wild type and PAM unwinding, activation and catalytic mutants. Oligonucleotides 3F-T-AAG-30 and 5F-NT-TTC-30 were used as substrates (Supplementary Table II). T-strand (TS) and NT-strand (NTS) products are marked. DNA markers are shown in nucleotides. e Quantification of the activity based on the cleavage experiments as shown in d. Bars represent mean ± SD. n > 3 independent experiments.