4UN5

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

4UN5 の概要

• エントリーDOI: 10.2210/pdb4un5/pdb
• 関連するPDBエントリー: 4UN3 4UN4
• 分子名称: SGRNA, CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1, TARGET DNA STRAND PROXIMAL FRAGMENT, ... (7 entities in total)
• 機能のキーワード: hydrolase-dna-rna complex, genome editing, rnp, protein-rna complex, hydrolase/dna/rna
• 由来する生物種: STREPTOCOCCUS PYOGENES; 詳細
• タンパク質・核酸の鎖数: 5
• 化学式量合計: 197779.31

構造登録者

Anders, C.,Niewoehner, O.,Duerst, A.,Jinek, M. (登録日: 2014-05-25, 公開日: 2014-07-23, 最終更新日: 2024-01-10)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2.4 Å)