適合活性化により,CRISPR-Cas12aの触媒とエンドヌクレアースの活性化のリセットが促進される

Stefano Stella¹, Pablo Mesa¹, Johannes Thomsen²

¹Structural Molecular Biology Group, Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.

Cell

|December 4, 2018

まとめ

CRISPR-Cas12a (Cpf1) 酵素について

科学分野:

分子生物学
生物化学
遺伝学

背景:

CRISPR-Cas12a (Cpf1) は,CRISPR-CasRNA誘導エンドヌクレアスの汎用性がある.
特定の二重鎖DNAの断裂を起こす能力のためにゲノム編集で広く使用されています

研究の目的:

Cas12aの触媒活性化とDNA分裂活動の構造的およびメカニズム的基礎を解明する.
Cas12aのSSDNA分裂のメカニズムとその調節を理解する.

主な方法:

クリオ電子顕微鏡 (cryo-EM) で,反応中間物質を視覚化する.
熱力学と運動学の研究のための単分子フォースター共振エネルギー転送 (smFRET).
DNAの分裂とcrRNAの移転を評価する生化学的測定法

主要な成果:

Cas12aの活性化を誘発するcrRNA-DNAハイブリッドを感知する3つの主要なタンパク質領域を特定した.
形状の変化は,フォスフォディエステル結合の水解と標的DNAの分裂につながることを示した.
Cas12aは無差別に ssDNAの分裂活動を活性化後に示しています
新しいcrRNAがRループを位移させ,SSDNAの劣化を終了させ,特異性をリセットすることを示した.

結論:

形状活性化が無差別なssDNA分裂を誘発するモデルを提案した.
crRNA媒介によるRループのシフトが,Cas12aの特異性をリセットするメカニズムであることを確認した.
精密なゲノム編集アプリケーションのためのCas12a活動の規制に関する洞察を提供しました.

キーワード:

CRISPR-Casについてクリオ・エムゲノム編集タンパク質-RNA-DNA複合体単分子FRET

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