Fig. 5: Assembly of the crRNA/DNA hybrid and activation of the RuvC pocket.

a View of the hybrid showing the interaction of the crRNA with residues in the RuvC insertion (Supplementary Fig. 5d). b Inset depicting the hydrophobic interaction between the turn of the RuvC insertion and the and cavity in the STP ___domain (Supplementary Fig. 5f). c 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. d Quantification of the activity based on the cleavage experiments as shown in c Bars represent the mean ± SD. n > 3 independent experiments. e Detailed view of the catalytic site containing a dinucleotide and a divalent metal. The D708 side chain and the associated distances are shown for visualization purposes only (see Supplementary Fig. 5f).