Structural basis for the activation of a compact CRISPR-Cas13 nuclease

Xiangyu Deng, Emmanuel Osikpa, Jie Yang, Seye J. Oladeji, Jamie Smith, Xue Gao () and Yang Gao ()
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Xiangyu Deng: Rice University
Emmanuel Osikpa: Rice University
Jie Yang: Rice University
Seye J. Oladeji: Rice University
Jamie Smith: Rice University
Xue Gao: Rice University
Yang Gao: Rice University

Nature Communications, 2023, vol. 14, issue 1, 1-12

Abstract: 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.

Date: 2023
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DOI: 10.1038/s41467-023-41501-5

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