Deng, XiangyuOsikpa, EmmanuelYang, JieOladeji, Seye J.Smith, JamieGao, XueGao, Yang2024-05-032024-05-032023Deng, X., Osikpa, E., Yang, J., Oladeji, S. J., Smith, J., Gao, X., & Gao, Y. (2023). Structural basis for the activation of a compact CRISPR-Cas13 nuclease. Nature Communications, 14(1), 5845. https://doi.org/10.1038/s41467-023-41501-5https://hdl.handle.net/1911/115610The 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.engExcept where otherwise noted, this work is licensed under a Creative Commons Attribution (CC BY) license. Permission to reuse, publish, or reproduce the work beyond the terms of the license or beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.Journal articles41467-023-41501-5https://doi.org/10.1038/s41467-023-41501-5