Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage

To protect against mobile genetic elements (MGEs), some bacteria and archaea have clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) adaptive immune systems. CRISPR RNAs (crRNAs) bound to Cas nucleases hybridize to MGEs based on sequence complementarity to guide the nucleases to cleave the MGEs. This programmable DNA cleavage has been harnessed for gene editing. Safety concerns include off-target and guide RNA (gRNA)-free DNA cleavages, both of which are observed in the Cas nuclease commonly used for gene editing, Streptococcus pyogenes Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9^H982A) devoid of gRNA-free DNA cleavage activity that is more selective for on-target cleavage. The H982A substitution in the metal-dependent RuvC active site reduces Mn²⁺-dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn²⁺, but not Mg²⁺, produces a gRNA-free DNA cleavage competent state that is disrupted by the H982A substitution. Our study demonstrates the feasibility of modulating cation:protein interactions to engineer safer gene editing tools.