Fig. 4. Engineered Cas9s improve specificity through a decreased rate of DNA cleavage.

a Schematic of DNA trap experiment. b, c SpCas9 cleavage of on-target (from Gong et al.¹⁵) (b) or off-target (c) DNA. d, e HypaCas9 cleavage of on-target (d) or off-target (e) DNA. f, g Cas9-HF1 cleavage of on-target (f) or off-target (g) DNA. Enzyme (28 nM) and labeled DNA (10 nM) were incubated in the absence of Mg²⁺ and the reaction was initiated by adding Mg²⁺ in the presence or absence of an excess of unlabeled DNA trap. A_• and A_◦ represent the amplitude with (filled circles) and without trap (open circles), respectively. The percentage indicates the fraction of pre-bound DNA committed to going forward for cleavage relative to the reaction in the absence of trap.