自然に発生するCRISPR-Cas9のオフスイッチ

April Pawluk¹, Nadia Amrani², Yan Zhang²

¹Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.

Cell

|December 17, 2016

まとめ

CRISPR-Cas9ゲノム編集を阻害する新しいアンチCRISPRタンパク質を発見しましたこれらの天然阻害剤は遺伝子編集技術の正確な制御に不可欠な"オフスイッチ"を提供する.

科学分野:

分子生物学
遺伝学
微生物学

背景:

CRISPR-Cas9の遺伝子編集は強力なツールを提供しますが精密な制御メカニズムには欠けています
バクテリオファージでコードされた抗CRISPRは以前も確認されていたが,タイプIのCRISPR- Casシステムでのみ確認された.
Cas9システムに対するアンチCRISPRの欠如は,条件付きゲノム編集のアプリケーションを制限しました.

研究の目的:

CRISPR-Cas9システムを阻害する新種の抗CRISPRタンパク質を発見する
新しく発見された抗CRISPRの抑制メカニズムを特徴づける.
CRISPR-Cas9に対する制御可能な"オフスイッチ"としてこれらのアンチCRISPRの有用性を実証する.

主な方法:

抗CRISPR候補者を特定するためのバイオ情報学的スクリーニングと機能的検査
NmeCas9に対する抗CRISPRの直接結合を確認するための生化学実験.
CRISPR-Cas9 ゲノム編集の抑制を検証するためにヒトの細胞ベースの測定法.

主要な成果:

Neisseria meningitidis Cas9 (NmeCas9) を標的にする3つの異なる抗CRISPRタンパク質ファミリーの発見
これらの抗CRISPRがNmeCas9に直接結合することを示す.
これらのタンパク質を用いてヒト細胞におけるNmeCas9媒介によるゲノム編集を成功裏に抑制した.

結論:

特定された抗CRISPRは,CRISPR- Cas9システムを特異的かつ強力に抑制する.
これらのタンパク質は遺伝的にコード化された"オフスイッチ"として機能し,条件付きCRISPR-Cas9の活性化を可能にします.
この発見は,真核生物におけるCRISPR-Cas9ゲノム編集の制御性と安全性を大幅に向上させます.

キーワード:

CRISPR-Casについてカス9 ネイセリア・メンニジティスアンチCRISPR ゲノム編集ファージ

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