2. Academic Validation
  3. Structural insights into the diversity and DNA cleavage mechanism of Fanzor

Structural insights into the diversity and DNA cleavage mechanism of Fanzor

  • Cell. 2024 Sep 19;187(19):5238-5252.e20. doi: 10.1016/j.cell.2024.07.050.
Peiyu Xu 1,  Makoto Saito 1,  Guilhem Faure 1,  Samantha Maguire 1,  Samuel Chau-Duy-Tam Vo 1,  Max E Wilkinson 1,  Huihui Kuang 2,  Bing Wang 2,  William J Rice 3,  Rhiannon K Macrae 1,  Feng Zhang 4
Affiliations

Affiliations

  • 1 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Cambridge, MA 02139, USA.
  • 2 Cryo-Electron Microscopy Core, NYU Grossman School of Medicine, New York, NY 10016, USA.
  • 3 Cryo-Electron Microscopy Core, NYU Grossman School of Medicine, New York, NY 10016, USA; Department of Cell Biology, NYU Grossman School of Medicine, New York, NY 10016, USA.
  • 4 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; McGovern Institute for Brain Research at MIT, Cambridge, MA 02139, USA; Department of Brain and Cognitive Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Howard Hughes Medical Institute, Cambridge, MA 02139, USA. Electronic address: zhang@broadinstitute.org.
PMID: 39208796 DOI: 10.1016/j.cell.2024.07.050
Abstract

Fanzor (Fz) is an ωRNA-guided Endonuclease extensively found throughout the eukaryotic domain with unique gene editing potential. Here, we describe the structures of Fzs from three different organisms. We find that Fzs share a common ωRNA interaction interface, regardless of the length of the ωRNA, which varies considerably across species. The analysis also reveals Fz's mode of DNA recognition and unwinding capabilities as well as the presence of a non-canonical catalytic site. The structures demonstrate how protein conformations of Fz shift to allow the binding of double-stranded DNA to the active site within the R-loop. Mechanistically, examination of structures in different states shows that the conformation of the lid loop on the RuvC domain is controlled by the formation of the guide/DNA heteroduplex, regulating the activation of nuclease and DNA double-stranded displacement at the single cleavage site. Our findings clarify the mechanism of Fz, establishing a foundation for engineering efforts.

Keywords

DNA cleavage mechanisms; Fanzor; R-loop structure; activation mechanisms; catalytic site; eukaryotic RNA-guided DNA endonuclease; gene editing; structural diversity.

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