Figure 1. Alt-R S.p. HiFi Cas9 Nuclease 3NLS provides consistently high on-target performance, while reducing off-target editing, when delivered as a ribonucleoprotein (RNP). RNP complexes (1 µM) were formed with wild-type Cas9 protein (Alt-R S.p. Cas9 Nuclease 3NLS; dark blue) and 3 high-fidelity Cas9 variants, Alt-R HiFi Cas9 (orange), SpCas9-HF1 (light blue; Kleinstiver et al., Nature 529:490–495), or eSpCas9 (gray; Slaymaker et al., Science 351:84–88), combined with an Alt-R crRNA:tracrRNA gRNA complex targeting the EMX1, HEKSite4, or VEGFA3 loci. Complexes (10 nM) were delivered into HEK-293 cells by reverse transfection using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher), and DNA was extracted after 48 hr. Editing was measured by PCR amplification of the indicated on- and off-target sites, followed by cleavage with T7EI and analysis using the Fragment Analyzer™ system (Advanced Analytical). Error bars represent the standard errors of the means. The sequence of the on- and off-target sites for each crRNA are indicated at the bottom (red = mismatch in protospacer; blue = mismatch in PAM site).