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TitleStructural basis for the activation of a compact CRISPR-Cas13 nuclease.
Journal, issue, pagesNat Commun, Vol. 14, Issue 1, Page 5845, Year 2023
Publish dateSep 20, 2023
AuthorsXiangyu Deng / Emmanuel Osikpa / Jie Yang / Seye J Oladeji / Jamie Smith / Xue Gao / Yang Gao /
PubMed AbstractThe CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 ...The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal "UC" sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications.
External linksNat Commun / PubMed:37730702 / PubMed Central
MethodsEM (single particle)
Resolution3.5 Å
Structure data

29433

8fti

EMDB-29433, PDB-8fti:
Cryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex in activated state
Method: EM (single particle) / Resolution: 3.5 Å

Source
  • planctomycetota bacterium (bacteria)
  • synthetic construct (others)
KeywordsRNA BINDING PROTEIN/RNA / Cas13bt3-crRNA-target RNA ternary complex / activated state / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex

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