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データを開く
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基本情報
登録情報 | データベース: PDB / ID: 7z4d | ||||||
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タイトル | Crystal structure of SpCas9 bound to a 10 nucleotide complementary DNA substrate | ||||||
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![]() | HYDROLASE / CRISPR / Cas9 / R-loop / substrate binding / off-target | ||||||
機能・相同性 | ![]() maintenance of CRISPR repeat elements / 3'-5' exonuclease activity / DNA endonuclease activity / defense response to virus / 加水分解酵素; エステル加水分解酵素 / DNA binding / RNA binding / metal ion binding 類似検索 - 分子機能 | ||||||
生物種 | ![]() synthetic construct (人工物) | ||||||
手法 | ![]() ![]() ![]() | ||||||
![]() | Pacesa, M. / Jinek, M. | ||||||
資金援助 | ![]()
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![]() | ![]() タイトル: R-loop formation and conformational activation mechanisms of Cas9. 著者: Martin Pacesa / Luuk Loeff / Irma Querques / Lena M Muckenfuss / Marta Sawicka / Martin Jinek / ![]() 要旨: Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise ...Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage. The programmable activity of Cas9 has been widely utilized for genome editing applications, yet its precise mechanisms of target DNA binding and off-target discrimination remain incompletely understood. Here we report a series of cryo-electron microscopy structures of Streptococcus pyogenes Cas9 capturing the directional process of target DNA hybridization. In the early phase of R-loop formation, the Cas9 REC2 and REC3 domains form a positively charged cleft that accommodates the distal end of the target DNA duplex. Guide-target hybridization past the seed region induces rearrangements of the REC2 and REC3 domains and relocation of the HNH nuclease domain to assume a catalytically incompetent checkpoint conformation. Completion of the guide-target heteroduplex triggers conformational activation of the HNH nuclease domain, enabled by distortion of the guide-target heteroduplex, and complementary REC2 and REC3 domain rearrangements. Together, these results establish a structural framework for target DNA-dependent activation of Cas9 that sheds light on its conformational checkpoint mechanism and may facilitate the development of novel Cas9 variants and guide RNA designs with enhanced specificity and activity. | ||||||
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-検証レポート
文書・要旨 | ![]() | 524.1 KB | 表示 | ![]() |
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-関連構造データ
関連構造データ | ![]() 7z4cC ![]() 7z4eC ![]() 7z4gC ![]() 7z4hC ![]() 7z4iC ![]() 7z4jC ![]() 7z4kC ![]() 7z4lC ![]() 5fq5S S: 精密化の開始モデル C: 同じ文献を引用 ( |
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類似構造データ | 類似検索 - 機能・相同性 ![]() |
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集合体
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非結晶学的対称性 (NCS) | NCSドメイン:
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