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CRISPR Guide RNA Cloning for Mammalian Systems
Published on: October 2, 2018
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CRISPR-Cas13dのRNA誘導リボヌクレアース活性に対する構造的基礎
Cheng Zhang1, Silvana Konermann2, Nicholas J Brideau2
1Laboratory of Genetics, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA; Helmsley Center for Genomic Medicine, Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA.
Cell
|September 23, 2018
まとめ
研究者は,CRISPR-Casシステムの重要な構成要素であるコンパクトなCas13d酵素の構造と機能を明らかにしました. これは,高度なトランスクリプトーム工学のためのRNA誘導RNAターゲティングの洞察を提供します.
科学分野:
- 分子生物学
- 生物化学
- 遺伝学
背景:
- CRISPR-Casシステムは,プロカリオットの適応免疫を提供し,遺伝子工学で広く使用されています.
- タイプVI-DのCRISPRシステムは,特定のRNA分子を標的と分割するためにガイドRNAを使用する小さな単一エフェクターCas13d酵素を備えています.
研究 の 目的:
- Cas13d機能の分子基礎とそのコンパクトな構造を理解する.
- Cas13dのRNA誘導,RNAターゲティングメカニズムについての洞察を提供するためです.
- 改善されたトランスクリプトーム工学技術の設計のための基盤を確立する.
主な方法:
- クリオ電子顕微鏡 (cryo-EM) を使用して,Cas13dガイドRNAバイナリおよびCas13dガイド-ターゲットRNA三重複合体の構造を解析した.
- 低解像度のアポ Cas13dの再構築は,冷凍-EMを用いて得られた.
- 水素-デュテリウム交換質量スペクトロメトリ (HDX-MS) を用いて構成動態を研究した.
- 機能的特徴化のために生化学的および細胞的測定を行った.
主要な成果:
- Cas13d複合体の高解像度の冷凍-EM構造は分子構造を明らかにした.
- アポ Cas13dとHDX-MSデータの6. 5 Åの再構築は,RNAスキャンに関連する構成動態を解明した.
- この研究は,Cas13dによるRNA誘導RNAターゲティングのメカニズムに関する詳細な洞察を提供します.
結論:
- 解明された構造とダイナミック情報は,Cas13dの機能の分子理解を提供します.
- これらの発見は,新しい,強化されたトランスクリプトームエンジニアリングツールの合理的な設計のための基礎を築いた.
- この研究は,遺伝子応用のためのRNAターゲティング技術の分野を前進させています.
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