a, Top: domain organization of AceCas9 shown as colored blocks in the direction from the N terminus to the C terminus. The regions corresponding to the structural domains are colored and labeled, and the relevant residues are labeled. RuvC-I–RuvC-III, discontinuous segments of the RuvC domain; BH, bridge helix; REC1, nucleic acid-recognition domain 1; REC2, nucleic acid-recognition domain 2; HNH, HNH nuclease domain; PID, PAM interaction domain. Bottom: schematic diagram of the nucleic acids used in this study, shown as nucleotides in the predicted secondary structures. Cleavage sites for the NTS DNA by the RuvC domain and the TS DNA by the HNH domain are indicated by the green and purple downtriangles, respectively. The PAM and the guide region are highlighted in gray.The TS and NTS are numbered sequentially with NTS numbers denoted with asterisks. b, Overlay of the gel filtration profiles of the AceCas9 protein and its ribonucleoprotein (RNP) complex assembled with the sgRNA shown in a. Samples collected for biochemistry and cryo-EM analysis are highlighted by the gray shaded area. c, Cleavage results of double-stranded DNA (dsDNA) assembled with either TS DNA labeled with hexachlorofluorescein (HEX) (red) or the NTS oligonucleotide labeled with fluorescein amidites (FAM) (green) at 10 nM by AceCas9 or its catalytic mutants at 1 μM in the presence of various divalent ions at 10 mM. WT, wild-type AceCas9; U, uncleaved DNA substrate; C cleaved DNA substrate; dHNH, AceCas9 with deactivated HNH; dRuvC, AceCas9 with deactivated RuvC. Credit: Nature Catalysis (2023). DOI: 10.1038/s41929-023-01031-1″>