進化したCas9変種,広範囲のPAM互換性と高いDNA特異性
Johnny H Hu1,2,3, Shannon M Miller1,2,3, Maarten H Geurts1,2,3
1Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.
Nature
|March 8, 2018
まとめ
科学者たちは,より広い範囲のDNA配列を認識する拡張されたCRISPR-Cas9変種 (xCas9) を開発しました. この突破はヒト細胞のゲノム編集能力と特異性を高めています
科学分野:
- 分子生物学
- 遺伝学
- バイオテクノロジー
背景:
- CRISPR-Cas9のゲノム編集は,プロトスペーサー隣接モチーフ (PAM) と呼ばれる特定のDNA配列の認識に依存しています.
- 広く使用されているStreptococcus pyogenes Cas9 (SpCas9) はNGG PAM配列に限定されています.
- 現存するCas9変種は哺乳類の細胞アプリケーションに限られたPAMの柔軟性を提供します.
研究 の 目的:
- 拡張されたPAM認識プロファイルを持つCas9の変種を設計する
- ヒトの細胞における 設計された変異体の機能性と特異性を評価する.
- CRISPR-Cas9技術の標的範囲を拡大する.
主な方法:
- 拡張されたPAM SpCas9変種 (xCas9) を進化させるために,ファージ支援連続進化 (PACE) が使用された.
- 機能的評価には,ヒト細胞における標的型転写活性化,遺伝子破壊,塩基編集が含まれていた.
- DNAの特異性を決定するために,全ゲノムのオフターゲットの活性が評価されました.
主要な成果:
- 進化したxCas9変種は,NG,GAA,GATを含む幅広いPAM配列を認識しています.
- xCas9は,哺乳類の細胞で活性化しているCas9タンパク質に対して報告された最も広範なPAM互換性を示しています.
- xCas9は,SpCas9と比較して,著しく高いDNA特異性と低いオフターゲットの活性を示しています.
結論:
- 設計されたxCas9はCRISPRシステムの DNAターゲティング能力を拡張します
- この変種は,人間の細胞での多様なCRISPRアプリケーションを,精度が向上した形でサポートしています.
- 拡張されたPAM互換性は,Cas9編集効率やDNA特異性とのトレードオフを必要としません.
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