The study reports the crystal structure of Streptococcus pyogenes Cas9 bound to a single-molecule guide RNA (sgRNA) and a target DNA containing a 5'-NGG-3' PAM. The structure reveals that the PAM motif is base-paired in a DNA duplex, with the GG dinucleotide in the non-complementary strand interacting with conserved arginine residues in 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 immediately upstream of the PAM. These findings suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. The study also establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities. Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. PAM recognition is critical for Cas9-mediated DNA targeting, as it is required for ATP-independent strand separation and guide RNA-target DNA heteroduplex formation. Recent crystal structures and electron microscopic reconstructions of Cas9 and its RNA- and DNA-bound complexes revealed that Cas9 undergoes a dramatic RNA-induced conformational rearrangement that facilitates target DNA binding. Although two tryptophan residues have been implicated in PAM binding, the molecular mechanism of PAM recognition remains unclear. To elucidate the molecular mechanism of PAM recognition in Cas9, the crystal structure of Streptococcus pyogenes Cas9 in complex with an 83-nucleotide sgRNA and a partially duplexed target DNA containing a 5'-TGG-3' PAM sequence was determined. The structure reveals an intact target DNA strand and a cleaved product that has dissociated from the RuvC domain active site. The bound nucleic acids are enclosed by the nuclease and helical recognition lobes of Cas9 and form a four-way junction that straddles the arginine-rich bridge helix. The entire PAM-containing region of the target DNA is base-paired, with strand separation occurring only at the first base pair of the target sequence. The PAM duplex is nestled in a positively charged groove between the Topo-homology and C-terminal domains. The deoxyribose-phosphate backbone of the non-target DNA strand is engaged in numerous ionic and hydrogen-bonding interactions. Conserved tryptophan residues Trp476 and Trp1126 are not in direct contact with the PAM, suggesting that the crosslinks may have originated from a transient intermediate in the PAM recognition mechanism. Instead, the guanine nucleobases of dG2* and dG3* in the non-target strand are read out in the major groove by base-specific hydrogen-bondThe study reports the crystal structure of Streptococcus pyogenes Cas9 bound to a single-molecule guide RNA (sgRNA) and a target DNA containing a 5'-NGG-3' PAM. The structure reveals that the PAM motif is base-paired in a DNA duplex, with the GG dinucleotide in the non-complementary strand interacting with conserved arginine residues in 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 immediately upstream of the PAM. These findings suggest a mechanism for PAM-dependent target DNA melting and RNA-DNA hybrid formation. The study also establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities. Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. PAM recognition is critical for Cas9-mediated DNA targeting, as it is required for ATP-independent strand separation and guide RNA-target DNA heteroduplex formation. Recent crystal structures and electron microscopic reconstructions of Cas9 and its RNA- and DNA-bound complexes revealed that Cas9 undergoes a dramatic RNA-induced conformational rearrangement that facilitates target DNA binding. Although two tryptophan residues have been implicated in PAM binding, the molecular mechanism of PAM recognition remains unclear. To elucidate the molecular mechanism of PAM recognition in Cas9, the crystal structure of Streptococcus pyogenes Cas9 in complex with an 83-nucleotide sgRNA and a partially duplexed target DNA containing a 5'-TGG-3' PAM sequence was determined. The structure reveals an intact target DNA strand and a cleaved product that has dissociated from the RuvC domain active site. The bound nucleic acids are enclosed by the nuclease and helical recognition lobes of Cas9 and form a four-way junction that straddles the arginine-rich bridge helix. The entire PAM-containing region of the target DNA is base-paired, with strand separation occurring only at the first base pair of the target sequence. The PAM duplex is nestled in a positively charged groove between the Topo-homology and C-terminal domains. The deoxyribose-phosphate backbone of the non-target DNA strand is engaged in numerous ionic and hydrogen-bonding interactions. Conserved tryptophan residues Trp476 and Trp1126 are not in direct contact with the PAM, suggesting that the crosslinks may have originated from a transient intermediate in the PAM recognition mechanism. Instead, the guanine nucleobases of dG2* and dG3* in the non-target strand are read out in the major groove by base-specific hydrogen-bond