5FW2

Crystal structure of SpCas9 variant EQR bound to sgRNA and TGAG PAM target DNA

5FW2 の概要

• エントリーDOI: 10.2210/pdb5fw2/pdb
• 関連するPDBエントリー: 5FW1 5FW3
• 分子名称: SGRNA, CRISPR-ASSOCIATED ENDONUCLEASE CAS9/CSN1, TARGET DNA STRAND, ... (7 entities in total)
• 機能のキーワード: hydrolase-dna-rna complex, crispr, cas9, endonuclease, pam, 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: 4
• 化学式量合計: 198422.66

構造登録者

Anders, C.,Bargsten, K.,Jinek, M. (登録日: 2016-02-11, 公開日: 2016-06-15, 最終更新日: 2024-01-10)

主引用文献

Anders, C.,Bargsten, K.,Jinek, M.
Structural Plasticity of Pam Recognition by Engineered Variants of the RNA-Guided Endonuclease Cas9.
Mol.Cell, 61:895-, 2016

PubMed Abstract: The RNA-guided endonuclease Cas9 from Streptococcus pyogenes (SpCas9) forms the core of a powerful genome editing technology. DNA cleavage by SpCas9 is dependent on the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) in the target DNA, restricting the choice of targetable sequences. To address this limitation, artificial SpCas9 variants with altered PAM specificities have recently been developed. Here we report crystal structures of the VQR, EQR, and VRER SpCas9 variants bound to target DNAs containing their preferred PAM sequences. The structures reveal that the non-canonical PAMs are recognized by an induced fit mechanism. Besides mediating sequence-specific base recognition, the amino acid substitutions introduced in the SpCas9 variants facilitate conformational remodeling of the PAM region of the bound DNA. Guided by the structural data, we engineered a SpCas9 variant that specifically recognizes NAAG PAMs. Taken together, these studies inform further development of Cas9-based genome editing tools.

PubMed: 26990992
DOI: 10.1016/J.MOLCEL.2016.02.020

実験手法

X-RAY DIFFRACTION (2.676 Å)