title: Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease.
abstract: 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 RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. 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.
authors: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors: Carolin
authors: C
authors: Anders
authors: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors: Ole
authors: O
authors: Niewoehner
authors: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors: Alessia
authors: A
authors: Duerst
authors: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.
authors: Martin
authors: M
authors: Jinek
mesh_terms: D001120:Arginine / Q000235:genetics / Q000378:metabolism; D020029:Base Pairing; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D008958:Models, Molecular; D009691:Nucleic Acid Denaturation; D059372:Nucleotide Motifs; D011487:Protein Conformation; D013297:Streptococcus pyogenes / Q000201:enzymology*; D013379:Substrate Specificity; D058727:RNA, Small Untranslated
chemical_list: D064130:CRISPR-Associated Proteins; D004247:DNA; D001120:Arginine; D004720:Endonucleases; D058727:RNA, Small Untranslated