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

Jonathan Gootenberg, Harvard University

Photo of Jonathan Gootenberg

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 <em>in vitro</em> 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 <em>Staphylococcus aureus</em>, SaCas9, which is small enough to allow for <em>in vivo</em> delivery.

Abstract Author(s): Jonathan Gootenberg, Aaron Smargon, Winston Yan, Ann Ran, Le Cong, Omar Abudayyeh, Eugene Koonin, Feng Zhang