Fig. 4: Activity of Cas9 orthologs at varying temperatures.

The cleavage activity of Cas9 orthologs was measured using in vitro DNA cleavage assays using fluorophore-labeled double-stranded DNA (dsDNA) substrates. Cleaved fragments were quantitated and are represented in a heatmap a showing overall activity at temperatures ranging from 10 °C to 68 °C. The intensity of the blue color indicates the proportion of substrate cleaved. Source data are provided in the Source Data file. b Cas9 orthologs with activity at elevated temperatures. In vitro DNA cleavage activity for a subset of Cas9 orthologs with >50% activity at 53 °C is summarized in a heatmap and plotted as the proportion of DNA substrate cleaved at varied temperatures. The intensity of the blue color in heatmaps indicates the proportion of substrate cleaved. Points represent the mean  ± SEM of at least three independent experiments. Green shading highlights the temperature range above 53 °C. c Cas9 orthologs with reduced activity at room temperature. In vitro DNA cleavage activity for a subset of Cas9 orthologs with <25% activity at 25 °C is summarized in a heatmap and plotted as a proportion of DNA substrate cleaved at varied temperatures. The intensity of the blue color in heatmaps indicates the proportion of substrate cleaved. Red shading highlights the temperature range below 25 °C Points represent the mean ± SEM of at least three independent experiments.