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TitleSequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition.
Journal, issue, pagesNucleic Acids Res, Vol. 54, Issue 1, Year 2026
Publish dateJan 5, 2026
AuthorsKazuki Ishihara / Shunsuke Matsumoto / Christoph Gerle / Chai C Gopalasingam / Hideki Shigematsu / Tsuyoshi Shirai / Tomoyuki Numata /
PubMed AbstractSome prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA- ...Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.
External linksNucleic Acids Res / PubMed:41495894 / PubMed Central
MethodsEM (single particle)
Resolution2.68 - 2.89 Å
Structure data

65338

9vtp

EMDB-65338, PDB-9vtp:
Target DNA-bound type I-F3 TniQ-Cascade of Vibrio parahaemolyticus in partial R-loop state
Method: EM (single particle) / Resolution: 2.68 Å

65339

9vtq

EMDB-65339, PDB-9vtq:
Target DNA-bound type I-F3 TniQ-Cascade of Vibrio parahaemolyticus in full R-loop state 1
Method: EM (single particle) / Resolution: 2.84 Å

65340

9vtr

EMDB-65340, PDB-9vtr:
Target DNA-bound type I-F3 TniQ-Cascade of Vibrio parahaemolyticus in full R-loop state 2
Method: EM (single particle) / Resolution: 2.89 Å

Chemicals

ChemComp-MG:
Unknown entry

Source
  • vibrio parahaemolyticus rimd 2210633 (bacteria)
  • vibrio alginolyticus (bacteria)
KeywordsDNA BINDING PROTEIN / CRISPR-associated transposon

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