フランセッラ・ノビシダの構造と工学 Cas9

Hisato Hirano¹, Jonathan S Gootenberg², Takuro Horii³

¹Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

Cell

|February 16, 2016

まとめ

研究者は,フランシセラ・ノビシダのCas9 (FnCas9) 構造を特徴付け,CRISPR-Cas9システムに保存され,異なる特徴を明らかにした. 彼らはより広いPAM配列を認識する変種を設計しゲノム編集機能を拡張しました

科学分野:

構造生物学
分子生物学
ゲノム工学

背景:

Cas9酵素はRNAによって誘導され,プログラム可能なゲノム編集のために二重鎖のDNA分裂を行います.
Cas9の活動は,特定のプロトスペーサー隣接モチーフ (PAM) 配列の要求によって制限されています.
Cas9-PAMの相互作用を理解することは,そのゲノム編集アプリケーションの拡大に不可欠です.

研究の目的:

ガイドRNAとDNAの標的と複合したFrancisella novicida Cas9 (FnCas9) の高解像度結晶構造を決定する.
FnCas9の構造を他のCas9オーソログと比較し,保存された特徴と異なる特徴を特定する.
変異したPAM特異性を有するFnCas9変異体を設計し,ゲノム編集におけるその有用性を実証する.

主な方法:

FnCas9ガイドRNA-DNA複合体の1.7 Å解像度構造を取得するために,X線結晶学を使用した.
FnCas9と他の既知のCas9オートログの構造を比較した.
サイト指向型変異は,PAM認識が修正されたFnCas9の変種を作成するために使用されました.
FnCas9-リボヌクレオプロテイン複合体のマイクロ注入は,マウスの生体ゲノム編集に使用された.

主要な成果:

結晶構造は,FnCas9の他のCas9のオートログと比較して保存され,異なる特徴を明らかにした.
FnCas9は5'-NGG-3' PAM配列を認識することが判明した.
緩和された5'-YG-3' PAM配列を認識するために変種FnCas9が設計されました.
マウスの卵巣にFnCas9を微量注入することで,5'- YG-3' PAMによる内生部位の編集が成功しました.

結論:

FnCas9に関する構造的な洞察は,CRISPR-Cas9システムの多様性をより深く理解します.
緩和されたPAM認識のためのFnCas9のエンジニアリングは,標的となるゲノムサイトの範囲を大幅に拡大します.
改良されたFnCas9システムは,哺乳類のシステムを含むゲノム編集におけるより広範な応用に期待されています.

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