Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNAs bearing sequences complementary to a 20-nucleotide segment in the guide RNA1,2. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control3. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA1,4-6. Here, we report a crystal structure of Streptococcus pyogenes Cas9 complexed with a single-molecule guide RNA (sgRNA) and a target DNA containing a canonical 5′-NGG-3′ PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major groove interactions with conserved arginine residues from the C-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation of the target DNA duplex immediately upstream of the PAM. These observations suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.