6AEG

Crystal structure of xCas9 in complex with sgRNA and target DNA (GAT PAM)

6AEG の概要

• エントリーDOI: 10.2210/pdb6aeg/pdb
• 分子名称: RNA (95-MER), DNA (25-MER), DNA (5'-D(*AP*AP*AP*GP*AP*TP*TP*AP*TP*TP*G)-3'), ... (5 entities in total)
• 機能のキーワード: enzyme, dna, nuclease, dna binding protein-dna-rna complex, dna binding protein/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: 202771.88

構造登録者

Guo, M.,Ren, K.,Zhu, Y.,Huang, Z. (登録日: 2018-08-04, 公開日: 2019-03-27, 最終更新日: 2023-11-22)

主引用文献

Guo, M.,Ren, K.,Zhu, Y.,Tang, Z.,Wang, Y.,Zhang, B.,Huang, Z.
Structural insights into a high fidelity variant of SpCas9.
Cell Res., 29:183-192, 2019

PubMed Abstract: The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9's off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.

PubMed: 30664728
DOI: 10.1038/s41422-018-0131-6

実験手法

X-RAY DIFFRACTION (2.701 Å)