Escherichia coliにおけるdCas9のターゲット検索の動態
Daniel Lawson Jones1, Prune Leroy1, Cecilia Unoson1
1Department of Cell and Molecular Biology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
Science (New York, N.Y.)
|October 1, 2017
まとめ
単一のCas9タンパク質が E. coliの標的DNA配列を見つけるのに6時間かかります 高濃度のCas9とガイドRNAは迅速なDNAターゲティングに必要である.
科学分野:
- 分子生物学
- 遺伝学
- 微生物学
背景:
- Cas9タンパク質はRNAによって導かれ 特定のDNA配列を標的にすることができます
- このプロセスは塩基配列を検証するために DNA の解き放たれを必要とします
- この探求のスピードを理解することは 遺伝子編集のアプリケーションにとって 極めて重要です
研究 の 目的:
- Cas9タンパク質が 特定の染色体DNA配列を 細胞内に特定するのにかかる時間を 決定する
- 触媒的に不活性なCas9 (dCas9) の細胞内検索メカニズムを調査する.
主な方法:
- 単分子光顕微鏡を用いた.
- 大量の制限-保護アッセイを使用した.
- 生きたEscherichia coliでdCas9を研究した.
主要な成果:
- 単一の光で標識されたdCas9タンパク質は 標的DNA配列を見つけるのに6時間かかります
- それぞれの潜在的標的DNA配列は 30ミリ秒未満で結合されます.
- 一旦結合すると,dCas9は複製されるまでDNAと結合し続けます.
- 高濃度のCas9とガイドRNAは,迅速なターゲティングに不可欠です.
結論:
- Cas9による特定のDNA配列の細胞内検索は 遅いプロセスです
- Cas9による効率的かつ迅速なDNAターゲティングは,タンパク質とガイドRNAの両方の高細胞内濃度を必要とする.
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