Fig. 5: dsDNA melting mechanisms by NTSB-containing PlmCas12e and NTSB-lacking VemCas12e and LesCas12e.

a, b Positively charged residues involved in DNA duplex interactions in the PlmCas12e NTSB (a PDB: 7WB1) and PlmCas12e NTSB (b, PDB: 7WAZ). The blue dashed lines indicate hydrogen-bonding interactions. c Comparisons of cis-cleavage on dsDNA by wild-type (WT) PlmCas12e and lysine residues-mutated PlmCas12e at varying salt concentrations. The cleaved fraction for each reaction was fit into the One Phase Decay model. k value is the rate constant. Data are shown as mean ± SD from n = 3 independent experiments. d Positively charged residues at corresponding positions to K148 and K150 within the Plm2Cas12e NTSB (predicted). e dsDNA cleavage efficiencies by wild-type Plm2Cas12e and arginine-restored Plm2Cas12e mutant at different salt concentrations. Data are shown as mean ± SD from n = 3 independent experiments. Statistical analysis was performed using a two-way ANOVA test with Sidak’s multiple comparison test. (****p value < 0.0001; “ns” means not significant with the p value > 0.05). The exact p values for the comparison at 25, 50, 100,150, 200, 300, and 400 mM NaCl are 0.9704, 0.8939, 0.1051, 0.9125, 0.1220, <0.0001, and 0.9953, respectively. Source data are provided as a Source Data file. f Comparison of structural elements of NTSB-containing PlmCas12e (PDB: 7WB1) and NTSB-lacking VemCas12e and LesCas12e. NTSB and Loop 1 are colored in purple, and Loop 2 is colored in brown. g Positively charged Loop 1 of VemCas12e. Four lysine residues are denoted. h The electron density map of Loop 2 among three Cas12e orthologs. Residues interacting with dsDNA duplex are denoted. i Schematic diagram showing different structural elements responsible for dsDNA invading in VemCas12e.