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CRISPR01:59

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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
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CRISPR-Cas9核酵素を設計し,ターゲット空間を拡大した

Hiroshi Nishimasu1, Xi Shi2,3, Soh Ishiguro4,5,6

  • 1Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. nisimasu@bs.s.u-tokyo.ac.jp nureki@bs.s.u-tokyo.ac.jp.

Science (New York, N.Y.)
|September 1, 2018
PubMed
まとめ

科学者は,リラックスしたNGプロトスペーサー隣接モチーフ (PAM) を認識するために,Cas9酵素変種 (SpCas9-NG) を設計した. これによって,ヒトの細胞内のこれまでアクセス不可能なゲノム位置をターゲットにすることで,ゲノム編集の能力を拡張します.

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科学分野:

  • 分子生物学
  • ゲノミクス
  • 生物化学

背景:

  • Cas9酵素はゲノム編集の重要なツールであり,標的のDNA分裂を可能にします.
  • 一般的に使用されているStreptococcus pyogenes Cas9 (SpCas9) 酵素は,特定のNGGプロトスペーサー隣接モチーフ (PAM) の要求によって制限されています.
  • このNGG PAMの特異性は,編集を対象とするゲノム部位の範囲を制限する.

研究 の 目的:

  • PAM認識機能の緩和された SpCas9 変種を設計する.
  • 野生型 SpCas9 の厳格なNGG PAM要求によって課される制限を克服するために.
  • CRISPRベースのゲノム編集のための標的ゲノムロキーの範囲を拡大する.

主な方法:

  • SpCas9酵素の合理的な工学により,変化したPAM特異性を有する変種 (SpCas9-NG) が作られる.
  • SpCas9-NGによるNG PAM認識の構造的基礎を決定するX線結晶学.
  • SpCas9-NGがNG PAM部位にインデルを誘発する能力を評価するためのヒト細胞における機能的測定.
  • SpCas9-NGと活性化誘発型シチジンデアミナーゼ (AID) の融合により,NG PAMサイトでの塩基編集能力をテストする.

主要な成果:

  • 緩和されたNG PAMを認識できる SpCas9-NG変種を成功裏に設計した.
  • SpCas9-NGは,変異した塩基特異の相互作用によってNG PAMに対応することを明らかにした.
  • SpCas9- NGは,ヒト細胞内の内生性NG PAM部位での挿入/削除 (インデル) を誘導する効果を示した.
  • SpCas9-NGがAIDと融合すると,ヒト細胞のNG PAM部位で標的型C-T変換が容易になりました.

結論:

  • SpCas9-NGは,Cas9ベースのゲノム編集技術の重要な進歩を表しています.
  • 設計されたSpCas9-NGは標的の範囲を拡大し,以前はアクセスできないゲノムロケーションで編集を可能にします.
  • AIDとSpCas9-NG融合は,NG PAMサイトで精密な塩基編集のための汎用ツールを提供し,ゲノムエンジニアリングアプリケーションを強化します.