Neither H982 nor H983 is placed on a flexible loop as is the histidine in the RNase H-type or the RNase T-type. The RuvC-type histidines will likely be able to abstract the proton and act as general base but not as third ion recruiter or facilitator of product release. However, there is a conserved lysine (Spy K974, Tth K112) and a conserved arginine (Spy R976) in close vicinity of the active site. This lysine (2-4 residues in direction of the N-terminus from the arginine) and the arginine are conserved among various Cas9 family members such as from Staphylococcus Aureus, Francisella Novicida, Actinomyces naeslundii and Campylobacter jejuni (Figure ). The lysine is very mobile (no sidechain density for Tth RuvC K112 PDB 4LD0) and most likely carries out a similar task such as K196 in Bh. In Gaussian Accelerated MD simulations of the activated Cas9 complex it has also been shown that R976 is more stable in a down conformation pointing in the active site. K196 in Bh stabilizes the negatively charged cleaved product until arrival of the third Mg 2+ which then possibly facilitates the product release. We postulate that one positively charged residue (K or R) is needed to facilitate catalysis and removal of the product in the RuvC-like type. Combinations of these general motifs are also possible such as in Bh RNase H. Here, the leaving group protonation most likely is RNase H-like, but the histidine, which is missing, is partly replaced by the substrate and transient metal ions and partly by the lysine as in the RuvC-like motif.