抗CRISPRタンパク質によるCRISPR-SpyCas9抑制の構造的基礎
De Dong1, Minghui Guo1, Sihan Wang1
1HIT Center for Life Sciences, School of Life Science and Technology, Harbin Institute of Technology, Harbin 150080, China.
Nature
|April 28, 2017
まとめ
2つのアンチCRISPRタンパク質は,PAMを模倣し,DNA結合を阻害することで,Cas9を阻害する. この構造的な洞察は,Cas9の発展を可能にします.
科学分野:
- 分子生物学
- 構造生物学
- 遺伝学
背景:
- CRISPR-Cas9システムは 細菌の免疫メカニズムで 外来DNAを標的にします
- Cas9酵素はガイドRNAを用いて特定の部位でDNAを割るため,プロトスペーサー付近モチーフ (PAM) が必要です.
- 抗CRISPRタンパク質AcrIIA2とAcrIIA4はCas9を阻害するが,そのメカニズムは不明である.
研究 の 目的:
- AcrIIA4がStreptococcus pyogenes Cas9 (SpyCas9) を抑制するメカニズムを解明する.
- 制御可能なCas9システムを開発するための構造的基盤を提供すること.
主な方法:
- シングルガイドRNA (sgRNA) とAcrIIA4を複合したSpyCas9のX線結晶学.
- Cas9-sgRNA-AcrIIA4複合体の構造分析と比較
主要な成果:
- AcrIIA4は sgRNAに依存した方法でSpyCas9と結合する.
- AcrIIA4はPAMを模倣し,DNA結合部位を遮断することで,Cas9を阻害する.
- また,AcrIIA4はCas9 RuvCの活性部位をシールドし,DNAの分裂を防ぐ.
- sgRNA結合は,SpyCas9のAcrIIA4結合部位の形成を誘導する.
結論:
- AcrIIA4は2つのメカニズムでSpyCas9を阻害する:DNA認識をブロックし,活性部位を遮断する.
- この発見は,CRISPR-Cas9遺伝子編集ツールの"オフスイッチ"メカニズムを設計するための構造的基盤を提供します.
- これらの相互作用を理解することは 精密なゲノム編集アプリケーションに不可欠です
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