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- Title
Structural basis for the activation of a compact CRISPR-Cas13 nuclease.
- Authors
Deng, Xiangyu; Osikpa, Emmanuel; Yang, Jie; Oladeji, Seye J.; Smith, Jamie; Gao, Xue; Gao, Yang
- Abstract
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. The authors present the structure of Cas13bt3 in an activated state, illustrating its target length-dependent activation mechanism, and use it as a guide to develop a high-fidelity Cas13bt3 variant suitable for RNA knockdown and therapeutic applications.
- Subjects
ADENO-associated virus; RIBONUCLEASES; RNA
- Publication
Nature Communications, 2023, Vol 14, Issue 1, p1
- ISSN
2041-1723
- Publication type
Article
- DOI
10.1038/s41467-023-41501-5