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CRISPR 適応による全ゲノムにわたる混乱は,抗生物質感受性の複雑な遺伝子を明らかにします
Wenyan Jiang1, Panos Oikonomou1, Saeed Tavazoie1
1Department of Biological Sciences, Columbia University, New York, NY 10027, USA; Department of Systems Biology, Columbia University, New York, NY 10032, USA; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
Cell
|February 29, 2020
まとめ
CRISPR適応媒介型ライブラリ製造 (CALM) は,細菌細胞から包括的なCRISPRiライブラリを作成し,コストを大幅に削減し,遺伝子機能の研究や抗生物質耐性メカニズムの発見に対する感度を増やす.
科学分野:
- 分子生物学
- 遺伝学
- 微生物学
背景:
- ゲノム全体のCRISPRスクリーンは遺伝子機能の研究には強力ですが,高価で低多様性のガイドRNAライブラリによって制限されています.
- ガイドRNAを合成する現在の方法は,高価で,時間がかかり,労働が密集しています.
研究 の 目的:
- 非常に多様なCRISPR干渉 (CRISPRi) ライブラリを生成するための新しい費用対効果の方法を開発する.
- 抗生物質耐性などの新しい遺伝的決定因子を特定するためのCRISPRスクリーニングの感度と範囲を高める.
主な方法:
- CRISPR適応媒介ライブラリ製造 (CALM) を開発するために,Streptococcus pyogenesのCRISPR-Cas適応装置を使用した.
- CALMは細菌の細胞を工場として利用し,95パーセントのゲノム部位を標的とした数十万の異なるcrRNAを生産する.
- 繰り返しのCRISPR適応は,二重遺伝子の混乱を研究するための二重crRNAライブラリを生成するために使用されました.
主要な成果:
- 遺伝子ごとに平均100以上の異なるcrRNAsを持つ非常に包括的なCRISPRiライブラリを生成し,微妙な転写抑制分析を可能にしました.
- 強化されたCRISPRiスクリーニングで新しい抗生物質耐性決定因子を発見しました.
- 繰り返しのCALMを用いて 10万以上の二重遺伝子の乱れを作り出し 抗生物質耐性の進化の歴史的な偶然性についての洞察を明らかにしました
- CALMは伝統的なgRNA合成とクローニングを回避し,野生型の細菌のライブラリ生成を可能にします.
結論:
- CALMは,多様なCRISPRiライブラリを生成するためのスケーラブルで費用対効果の高いアプローチを提供します.
- この方法によりCRISPRスクリーンの感度と適用性が著しく向上し,特に複雑な細菌系での遺伝子発見に役立ちます.
- CALMは,抗生物質耐性の段階的な獲得などの複雑な遺伝子相互作用と進化過程の研究を容易にする.
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