CRISPR-Cas proteins are present in many bacterial species to protect them from viruses. Their adaptation for use in genome editing of animal and plant species has revolutionized biology and medicine. The most commonly used CRISPR-Cas system is CRISPR-Cas9 from the bacterium Streptococcus pyogenes. However, sensitivity to inactivation limits its usefulness under certain conditions, particularly within cultured mammalian cells. It was hypothesized that Cas9 from an alternate bacterium, Geobacillus stearothermophilus (GeoCas9), would have more robust genome editing ability in cultured cells due to its increased thermal stability. Native GeoCas9 proved to be a poor genome editor when used in mammalian cells. However, sequence alterations to GeoCas9 resulted in a modified version, iGeoCas9, that was 100 fold more active in genome editing within mammalian cells. iGeoCas9 protein structure was studied to discover the structural basis of this improved activity. Similar modifications made to Cas9 proteins from other bacteria also showed increased genome editing in mammalian cells, demonstrating a way to enhance genome editing efficiency of CRISPR-Cas9 system.