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生体細胞におけるCRISPR-Cas9ゲノム調査の動態

Spencer C Knight¹, Liangqi Xie², Wulan Deng³

¹Department of Chemistry, University of California, Berkeley, CA, USA.

Science (New York, N.Y.)

|November 14, 2015

まとめ

CRISPRに関連するタンパク質Cas9は 3D拡散を使って哺乳類の細胞内のDNAターゲットを探しますその動きとサンプリングは局所的なクロマチンの環境によって影響を受けゲノム尋問メカニズムを明らかにします

科学分野:

分子生物学
遺伝学
生物化学

背景:

CRISPRに関連したタンパク質 Cas9 (CRISPR-Cas9) は,ゲノム編集,転写変調,および生細胞イメージングのための強力なツールです.
CRISPR-Cas9複合体は,RNA-DNA互補性を介して二重鎖DNAを標的とするが,哺乳類の細胞における体内検索メカニズムは不明である.

研究の目的:

哺乳類の細胞におけるCas9による標的探求の in vivoメカニズムを調査する.
生体細胞におけるCas9の拡散と染色体結合のダイナミクスを視覚化する.

主な方法:

生体細胞におけるCas9の動きとクロマチンの相互作用をモニタリングするために,単粒子の追跡を用いた.
分析は,拡散パターンと非標的結合イベントの持続時間に焦点を当てた.

主要な成果:

Cas9は主に哺乳類の細胞内のターゲット検索のために3次元拡散を使用します.
標的外結合は一時的なもので,平均して1秒未満です.
Cas9の移動とクロマチンの採取はヘテロクロマチンの領域に制限されており,環境依存性を示しています.

結論:

この研究では,真核細胞における細菌のCas9のインビボゲノム尋問戦略を明らかにした.
Cas9は,局所的なクロマチンの構造の影響で,クロマチンのアクセシビリティと拡散をバランスすることで,哺乳類のゲノムをナビゲートします.

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