7S37

Cas9:sgRNA (S. pyogenes) in the open-protein conformation

7S37 の概要

• エントリーDOI: 10.2210/pdb7s37/pdb
• 関連するPDBエントリー: 7S36 7S38 7S3H
• EMDBエントリー: 24817 24818 24819 24823
• 分子名称: CRISPR-associated endonuclease Cas9/Csn1, Single-guide RNA (2 entities in total)
• 機能のキーワード: dnase, complex, ribonucleoprotein, genome editor, hydrolase-rna complex, hydrolase/rna
• 由来する生物種: Streptococcus pyogenes serotype M1; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 191759.45

構造登録者

Cofsky, J.C.,Soczek, K.M.,Knott, G.J.,Nogales, E.,Doudna, J.A. (登録日: 2021-09-04, 公開日: 2022-04-20, 最終更新日: 2024-06-05)

主引用文献

Cofsky, J.C.,Soczek, K.M.,Knott, G.J.,Nogales, E.,Doudna, J.A.
CRISPR-Cas9 bends and twists DNA to read its sequence.
Nat.Struct.Mol.Biol., 29:395-402, 2022

PubMed Abstract: In bacterial defense and genome editing applications, the CRISPR-associated protein Cas9 searches millions of DNA base pairs to locate a 20-nucleotide, guide RNA-complementary target sequence that abuts a protospacer-adjacent motif (PAM). Target capture requires Cas9 to unwind DNA at candidate sequences using an unknown ATP-independent mechanism. Here we show that Cas9 sharply bends and undertwists DNA on PAM binding, thereby flipping DNA nucleotides out of the duplex and toward the guide RNA for sequence interrogation. Cryogenic-electron microscopy (cryo-EM) structures of Cas9-RNA-DNA complexes trapped at different states of the interrogation pathway, together with solution conformational probing, reveal that global protein rearrangement accompanies formation of an unstacked DNA hinge. Bend-induced base flipping explains how Cas9 'reads' snippets of DNA to locate target sites within a vast excess of nontarget DNA, a process crucial to both bacterial antiviral immunity and genome editing. This mechanism establishes a physical solution to the problem of complementarity-guided DNA search and shows how interrogation speed and local DNA geometry may influence genome editing efficiency.

PubMed: 35422516
DOI: 10.1038/s41594-022-00756-0

実験手法

ELECTRON MICROSCOPY (3.2 Å)