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TitleVisualization of a multi-turnover Cas9 after product release.
Journal, issue, pagesNat Commun, Vol. 16, Issue 1, Page 5681, Year 2025
Publish dateJul 1, 2025
AuthorsKaitlyn A Kiernan / David W Taylor /
PubMed AbstractWhile the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product ...While the most widely used CRISPR-Cas enzyme is the Cas9 endonuclease from Streptococcus pyogenes (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Cas9 can eventually be removed from the product by extrinsic factors, such as translocating polymerases, but the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and promote faster enzyme turnover. Using kinetics-guided cryo-EM, we examine the conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.
External linksNat Commun / PubMed:40593576 / PubMed Central
MethodsEM (single particle)
Resolution2.76 - 3.1 Å
Structure data

47834

9eak

EMDB-47834, PDB-9eak:
SpCas9 with 17-bp R-loop containing 2 terminal mismatches (State I - pre-activation)
Method: EM (single particle) / Resolution: 2.76 Å

47835

9eal

EMDB-47835, PDB-9eal:
SpCas9 with 17-bp R-loop containing 2 terminal mismatches (State II - precleavage)
Method: EM (single particle) / Resolution: 2.83 Å

47941

9ed9

EMDB-47941, PDB-9ed9:
SpCas9 with 17-bp R-loop containing 2 terminal mismatches (State III - checkpoint)
Method: EM (single particle) / Resolution: 2.95 Å

47942

9eda

EMDB-47942, PDB-9eda:
SpCas9 with 17-bp R-loop containing 2 terminal mismatches (State IV - product)
Method: EM (single particle) / Resolution: 2.88 Å

47943

9edb

EMDB-47943, PDB-9edb:
SpCas9 with 17-bp R-loop containing 2 terminal mismatches (State V - dissociated)
Method: EM (single particle) / Resolution: 3.1 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-HOH:
WATER

Source
  • streptococcus pyogenes (bacteria)
  • synthetic construct (others)
KeywordsHydrolase/DNA/RNA / CRISPR-associated endonuclease / DNA BINDING PROTEIN / Hydrolase-DNA-RNA complex / Type II-A / RNA-guided / DNA targeting / Mg2+ dependent / DNA BINDING PROTEIN/DNA/RNA / DNA BINDING PROTEIN-DNA-RNA complex

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