細胞型特異的な遺伝子編集のためのCRISPR-Cas9エンドヌクレアスの受容体媒介配送
Romain Rouet, Benjamin A Thuma1, Marc D Roy2
1Pfizer Medicine Design , Groton , Connecticut 06340 , United States.
Journal of the American Chemical Society
|April 19, 2018
まとめ
研究者はCRISPR-Cas9遺伝子編集タンパク質を 特定の細胞を標的とするリガンドで設計した. この受容体媒介による配送は,肝細胞の精密な遺伝子編集を 厳しい方法なしに可能にし, in vivo 応用への道を開きます.
科学分野:
- 分子生物学
- 遺伝学
- バイオテクノロジー
背景:
- CRISPR-Casシステムは 精密なゲノム編集が可能ですが ターゲットを絞った配送方法がありません
- CRISPRの研究と臨床応用を進めるには 細胞と組織の選択的な配送が不可欠です
研究 の 目的:
- 標的細胞の吸収のためにアシアログリコプロテイン受容体リガンド (ASGPrL) で設計されたCRISPR-Cas9タンパク質の設計と評価.
- 細胞型特異的受容体媒介遺伝子編集を in vitro で実証する.
主な方法:
- ASGPrリガンド (Cas9-ASGPrL RNP) を含んだStreptococcus pyogenes Cas9タンパク質をエンジニアリングした.
- 肝臓の細胞系 (HEPG2とSKHEP) の生細胞イメージングのために,光ラベル付きCas9-ASGPrL RNPを使用した.
- エンドソモリチスペプチドの存在で遺伝子編集の効率を評価した.
主要な成果:
- Cas9-ASGPrL RNPは,ASGPrを発現するHEPG2細胞に,対照細胞であるSKHEP細胞に比べて優位な内部化を示した.
- 有効なASGPr媒介の内細胞化と標的細胞におけるRNPの蓄積が示されている.
- 細胞型特異的受容器誘導の遺伝子編集が,電解または変異反応剤なしで達成された.
結論:
- ゲノム編集酵素を受容体媒介で提供することは,細胞選択的な遺伝子編集のための有効な戦略です.
- このアプローチは,肝選別遺伝子編集を in vivo で開発するための基礎を提供します.
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