CRISPR-Cas9によるDNA標的分裂の適合制御

Samuel H Sternberg¹, Benjamin LaFrance², Matias Kaplan³

¹Department of Chemistry, University of California, Berkeley, California 94720, USA.

Nature

|November 3, 2015

まとめ

Cas9 (CRISPR関連タンパク質9) のDNA分裂は,HNH核酸ドメインによって制御される.

科学分野:

分子生物学
生物化学
遺伝学

背景:

Cas9は,バクテリアのCRISPR適応免疫の中心にあるRNA誘導DNA内核酶である.
シングルガイドRNAを持つCas9は,様々な生物におけるゲノム工学の重要なツールです.
Cas9のDNA分裂特異性を理解することは,治療用途において極めて重要です.

研究の目的:

Cas9のDNA分裂活動を制御する分子メカニズムを解明する.
DNAターゲティングにおけるHNH核酸ドメインの構成の役割を調査する.
最初のDNA結合を超えて,Cas9の特異性を制御する要因を特定する.

主な方法:

分子内フォースター共振エネルギー伝送 (FRET) 実験を活用した.
対象DNAと対象外DNAのCas9触媒ドメインの相対的方向性を測定した.
HNH核酸ドメインの構成状態を分析した.

主要な成果:

Cas9 DNAの分裂効率は,HNHドメインの活性化構造のサンプリングと相関する.
新しいアロステル伝達メカニズムは,Cas9核酵素ドメインの両方の調整された活性化を保証する.
DNAの分裂は,PAM認識とRNA-DNA塩基配列を超えた校正メカニズムによって制御される.

結論:

HNH核酸ドメインの構成状態は,Cas9 DNAの分裂の直接的決定因子である.
Cas9は,最終的な校正ステップを含む,多層の特異性チェックポイントを持っています.
これらの発見は,Cas9の機能の理解を深め,より正確なゲノム編集ツールの開発を促します.

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