Characterization of CRISPR Systems With High-throughput Sequencing to Diversify the Molecular Toolset

The Type II CRISPR system encodes an intriguing RNA-guided DNA nuclease Cas9. The programmability of Cas9 has resulted in its wide adoption as a genome editing tool. However, very few of the known Cas9 orthologs have been characterized and developed as alternative tools for easier delivery, orthogonality, or more specific editing. Using a combination of in vitro cutting assays and next-generation sequencing of bacterial hosts, we have characterized new Cas9 orthologs and their associated guide RNAs. Characterized orthologs include the small Cas9 from Staphylococcus aureus, SaCas9, which is small enough to allow for in vivo delivery.