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

Crystal structure of the RNA-guided endonuclease Cas9 bound to a guide RNA and a target DNA duplex reveals how base-specific recognition of a short motif known as PAM in the DNA target results in localized strand separation in the DNA immediately upstream of the PAM, allowing the target DNA strand to hybridize to the guide RNA. The bacterial Cas9 nuclease is an RNA-guided DNA endonuclease found in bacterial CRISPR defence systems and is also widely used as a genetic engineering tool. Cas9 associates with a guide RNA, forms a complex with complementary duplex DNA, and cleaves the DNA. For cleavage to occur, the DNA target must contain a trinucleotide motif known as PAM. Martin Jinek and colleagues have solved the structure of Cas9 bound to a guide RNA and a PAM-containing duplex DNA. The structure reveals how base-specific recognition of PAM in the DNA target results in localized strand separation in the DNA immediately upstream of the PAM, allowing the target DNA strand to hybridize to the guide RNA. The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA 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 DNA1,4,5,6. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA 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 carboxy-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 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.