R-loop formation and conformational activation mechanisms of Cas9.
Nature
; 609(7925): 191-196, 2022 09.
Article
em En
| MEDLINE
| ID: mdl-36002571
ABSTRACT
Cas9 is a CRISPR-associated endonuclease capable of RNA-guided, site-specific DNA cleavage1-3. The programmable activity of Cas9 has been widely utilized for genome editing applications4-6, 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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Base de dados:
MEDLINE
Assunto principal:
Streptococcus pyogenes
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Microscopia Crioeletrônica
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Sistemas CRISPR-Cas
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Domínios Proteicos
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Proteína 9 Associada à CRISPR
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Estruturas R-Loop
Idioma:
En
Revista:
Nature
Ano de publicação:
2022
Tipo de documento:
Article
País de afiliação:
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