State Key Laboratory of Genetic Engineering
School of Life Sciences, Fudan University
(Background) The CRISPR/Cas9 system provides unprecedented genome editing capabilities, but it requires a protospacer adjacent motif (PAM) for target sequence binding. Previous PAM screening approaches are performed in vitro or in bacteria. (Methods) Here, we developed a GFP-based approach for PAM screening in human cells. This approach is highly sensitive that can even detect PAMs associated with single cleavage event. (Results) We use this approach to systematically evaluate PAMs recognized by SpCas9, and identified multiple alternative PAMs. In addition, we show that this approach enables to identify new CRISPR/Cas9 system for genome editing. We identified a small Cas9 orthologue that recognizes a simple PAM. (Conclusions) In summary, we offer a powerful platform for PAM screen and new Cas9 nuclease identification.