Thread by @zhangf

Since the discovery of CRISPR-Cas systems, people have wondered if a similar type of system exists in eukaryotes. Today, we report in @Nature the characterization of Fanzor, a eukaryotic RNA-guided system that can be reprogrammed for human genome editing. nature.com/articles/s4158…

1/x In exploring the evolution of Cas9, we identified Fanzor (Fz) as a putative RNA-guided nuclease while studying IscB, IsrB, and TnpB–collectively called OMEGA systems–in collaboration with Eugene Koonin’s group. Here, we expand our analysis of the diversity of Fz proteins,… https://t.co/7XIcScOPlhtwitter.com/i/web/status/1…

2/x Fz1 and Fz2 share similar domain architecture and catalytic residues with the OMEGA effector TnpB and the CRISPR effector Cas12, and our analysis suggests TnpB evolved into both prokaryotic Cas12 and eukaryotic Fz.

3/x Based on this, we predicted Fz is also an RNA-guided DNA endonuclease. To test this, we first performed RNA pull-downs with Fz1 and Fz2 and found these proteins bind to the non-coding RNA found in Fz loci, confirming this region encodes an ωRNA.

4/x We next experimentally confirmed that Fz1 and Fz2 perform ωRNA-guided DNA cleavage in a TAM-dependent manner, and obtained four active Fz orthologs (from fungus, algae, amoeba and hard clam).

5/x We then showed that Fz1 and Fz2 can be reprogrammed to target the human genome in cells.

6/x To boost in vivo editing, we focused on Fz1 from the fungus Spizellomyces punctatus (SpuFz1), and optimized both the ωRNA and protein component, achieving a nearly 10-fold increase in activity (max. 18.4% indel formation) on the human genome.

7/x Finally, to gain insight into the mechanism of Fz and guide future development, we used cryo-EM to resolve the structure of SpuFz1 in complex with ωRNA and target DNA at 2.7 Å.

8/x Fz shares structural similarities with TnpB and Cas12, but the interaction between the ωRNA and the catalytic domains of Fz is notably more extensive, suggesting that the ωRNA might play a role in the catalytic reactions involving Fz.

9/x This work was led by postdocs Makoto Saito and Peiyu Xu in collaboration with @GuilhemFaure , S. Maguire, @soumya_kannan12, @altaetran, S. Vo & A. Desimone. Congratulations on a beautiful and comprehensive study!