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Structure paper

TitleR-loop formation and conformational activation mechanisms of Cas9.
Journal, issue, pagesNature, Vol. 609, Issue 7925, Page 191-196, Year 2022
Publish dateAug 24, 2022
AuthorsMartin Pacesa / Luuk Loeff / Irma Querques / Lena M Muckenfuss / Marta Sawicka / Martin Jinek /
PubMed AbstractCas9 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.
External linksNature / PubMed:36002571 / PubMed Central
MethodsEM (single particle) / X-ray diffraction
Resolution2.54 - 4.14 Å
Structure data

EMDB-14493, PDB-7z4c:
SpCas9 bound to 6 nucleotide complementary DNA substrate
Method: EM (single particle) / Resolution: 3.87 Å

EMDB-14494, PDB-7z4e:
SpCas9 bound to 8-nucleotide complementary DNA substrate
Method: EM (single particle) / Resolution: 4.14 Å

EMDB-14496, PDB-7z4g:
SpCas9 bound to 12-nucleotide complementary DNA substrate
Method: EM (single particle) / Resolution: 3.64 Å

EMDB-14497, PDB-7z4h:
SpCas9 bound to 14-nucleotide complementary DNA substrate
Method: EM (single particle) / Resolution: 3.49 Å

EMDB-14498, PDB-7z4i:
SpCas9 bound to 16-nucleotide complementary DNA substrate
Method: EM (single particle) / Resolution: 3.12 Å

EMDB-14499, PDB-7z4j:
SpCas9 bound to 18-nucleotide complementary DNA substrate in the catalytic state
Method: EM (single particle) / Resolution: 2.99 Å

EMDB-14500, PDB-7z4k:
SpCas9 bound to 10-nucleotide complementary DNA substrate
Method: EM (single particle) / Resolution: 3.81 Å

EMDB-14501, PDB-7z4l:
SpCas9 bound to 18-nucleotide complementary DNA substrate in the checkpoint state
Method: EM (single particle) / Resolution: 2.54 Å

PDB-7z4d:
Crystal structure of SpCas9 bound to a 10 nucleotide complementary DNA substrate
Method: X-RAY DIFFRACTION / Resolution: 3.1 Å

Chemicals

ChemComp-K:
Unknown entry

ChemComp-HOH:
WATER

ChemComp-MG:
Unknown entry

Source
  • streptococcus pyogenes (bacteria)
  • synthetic construct (others)
KeywordsHYDROLASE / CRISPR / Cas9 / R-loop / substrate binding / off-target

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