Directed Evolution of Targeted Cas9 to Specific Cleavage Sites

CRISPR/Cas9 has wide-reaching scientific and therapeutic applications, but efficient, exact targeting of cutting sites can be challenging. Several cas9 variants have been engineered to target alternate PAMs; however, generation of new variants is often laborious. Additionally, the cutting ability of cas9 nucleases is often highly guide-dependent and shows a large amount of variation between sites. In order to engineer new nuclease variants targeted to cleave a given site, we developed a directed evolution platform to select for DNA cleavage in bacteria. Our system allows rapid generation of mutant libraries, which are challenged with phage containing target sites in a competitive pool. Using this method, we developed S. aureus cas9 variants which have altered PAM preferences to target cleavage to novel targets of therapeutic relevance.