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TitleCryo-EM structure of the RfxCas13d-crRNA-off-target-RNA complex.
Journal, issue, pagesStructure, Vol. 34, Issue 1, Page 11-19.e2, Year 2026
Publish dateJan 8, 2026
AuthorsQianxi Yang / Yifang Sun / Lei Sun / Tian Chi / Zhenguo Chen /
PubMed AbstractThe CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ...The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ribonuclease and has been engineered for programmable RNA editing, which is a commonly used, active, and well-characterized small type VI editor. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ruminococcus flavefaciens Cas13d in a RfxCas13d-crRNA-off-target-RNA ternary complex and RfxCas13d-crRNA binary complex at 3.10 and 3.13 Å resolution. The ternary complex consists of RfxCas13d, crRNA, and a captured short off-target ssRNA at a complex state of binding proximal mismatched RNA. RfxCas13d undergoes conformational changes with or without the off-target RNA, but the catalytic sites remain unchanged. Mg aids in stabilizing the crRNA repeat region structure, which may be crucial for RNA binding. This discovery provides the foundation for developing RfxCas13d as a mature tool and offers a framework for advancing transcriptome engineering.
External linksStructure / PubMed:41135510
MethodsEM (single particle)
Resolution3.1 Å
Structure data

63530

9lzt

EMDB-63530, PDB-9lzt:
RfxCas13d-crRNA binary complex
Method: EM (single particle) / Resolution: 3.1 Å

63531

9lzu

EMDB-63531, PDB-9lzu:
RfxCas13d-crRNA-target RNA ternary complex
Method: EM (single particle) / Resolution: 3.1 Å

Chemicals

ChemComp-MG:
Unknown entry

Source
  • Escherichia coli (E. coli)
  • cloning vector pgrna-ccdb-1 (others)
  • ruminococcus sp. xpd3002 (bacteria)
KeywordsRNA BINDING PROTEIN/RNA / Cas13d / RNA / Complex / RNA BINDING PROTEIN-RNA complex

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