Cas9 orthologs for genome and epigenome editing

CRISPR/Cas9 systems have revolutionized biotechnology, creating diverse new opportunities for biomedical research and therapeutic genome and epigenome editing. Despite the abundance of bacterial CRISPR/Cas9 systems, few are effective in human cells, limiting the overall potential impact of CRISPR technology. To expand CRISPR/Cas toolbox, we tested a set of CRISPR/Cas9 systems from bacterial species not know to routinely infect humans. Four systems demonstrated robust, specific gene repression in human cells when used as nuclease-null dCas9s fused with a KRAB domain and were also highly active nucleases in human cells. Three of the systems have distinct protospacer adjacent motifs (PAMs) compared to the commonly used S. aureus and S. pyogenes Cas9s. Additionally, we assessed gene activation when fused with the p300 catalytic domain. Notably, S. uberis Cas9 performed comparably to the widely used S. aureus and S. pyogenes Cas9s in repression, activation, and nuclease activity. This study expands the CRISPR/Cas9 repertoire, addressing key challenges in advancing effective genome and epigenome editing for diverse applications.