4UN5

Crystal structure of Cas9 bound to PAM-containing DNA target containing mismatches at positions 1-3

Summary for 4UN5

• Entry DOI: 10.2210/pdb4un5/pdb
• Related: 4UN3 4UN4
• Descriptor: SGRNA, CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1, TARGET DNA STRAND PROXIMAL FRAGMENT, ... (7 entities in total)
• Functional Keywords: hydrolase-dna-rna complex, genome editing, rnp, protein-rna complex, hydrolase/dna/rna
• Biological source: STREPTOCOCCUS PYOGENES; More
• Total number of polymer chains: 5
• Total formula weight: 197779.31

Authors

Anders, C.,Niewoehner, O.,Duerst, A.,Jinek, M. (deposition date: 2014-05-25, release date: 2014-07-23, Last modification date: 2024-01-10)

Primary citation

Anders, C.,Niewoehner, O.,Duerst, A.,Jinek, M.
Structural Basis of Pam-Dependent Target DNA Recognition by the Cas9 Endonuclease
Nature, 513:569-, 2014

PubMed 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.

PubMed: 25079318
DOI: 10.1038/NATURE13579

Experimental method

X-RAY DIFFRACTION (2.4 Å)