The Cas9 endonuclease is guided to its targets by single guide RNAs (sgRNAs). Looking to improve the DNA targeting-specificity of Cas9, Slaymaker et al. hypothesized that DNA strand separation enhances Cas9 nuclease activity, and therefore that inhibiting Cas9 helicase activity would reduce the strength of sgRNA–off-target-DNA interactions and thus decrease off-target cleavage. To test this, they generated Streptococcus pyogenes Cas9 (SpCas9) mutants at 32 positively charged residues within a SpCas9 groove, which is likely to support strand separation, and found 3 mutation combinations that retained SpCas9 wild-type on-target activity with undetectable off-target cleavage at selected genes. These SpCas9 mutants exhibited increased sensitivity to mismatches between sgRNA and target DNA, and genome-wide reduction in off-target cleavage levels.