5B2T

Crystal structure of the Streptococcus pyogenes Cas9 VRER variant in complex with sgRNA and target DNA (TGCG PAM)

5B2T の概要

• エントリーDOI: 10.2210/pdb5b2t/pdb
• 関連するPDBエントリー: 5B2R 5B2S
• 分子名称: Guide RNA, CRISPR-associated endonuclease Cas9, Target DNA, ... (9 entities in total)
• 機能のキーワード: crispr-cas9, genome engineering, hydrolase-rna-dna complex, hydrolase/rna/dna
• 由来する生物種: Streptococcus pyogenes serotype M1; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 197056.21

構造登録者

Hirano, S.,Nishimasu, H.,Ishitani, R.,Nureki, O. (登録日: 2016-02-02, 公開日: 2016-03-23, 最終更新日: 2023-11-08)

主引用文献

Hirano, S.,Nishimasu, H.,Ishitani, R.,Nureki, O.
Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9
Mol.Cell, 61:886-894, 2016

PubMed Abstract: The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9.

PubMed: 26990991
DOI: 10.1016/j.molcel.2016.02.018

実験手法

X-RAY DIFFRACTION (2.2 Å)