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bioRxiv · 10.1101/2021.06.27.449965

Structure of the mini-RNA-guided endonuclease CRISPR-Cas{Phi}3

Carabias del Rey, A.·Fuglsang, A.·Temperini, P.·Pape, T.·Sofos, N.·Stella, S.·Erlendsson, S.·Montoya, G.
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Abstract

CRISPR-Cas{Phi} is a novel family of miniaturized RNA-guided endonucleases from phages 1,2. These novel ribonucleoproteins (RNPs) provide a compact scaffold gathering all key activities of a genome editing tool2. Here, we provide the first structural insight into Cas{Phi} singular DNA targeting and cleavage mechanism by determining the cryoEM structure of Cas{Phi}3 with the triple strand R-loop generated after DNA cleavage. The structure reveals the unique machinery for target unwinding to form the crRNA-DNA hybrid and cleaving the target DNA. The protospacer adjacent motif (PAM) is recognised by the target strand (T-strand) and non-target strand (NT-strand) PAM interacting domains (TPID and NPID). Unwinding occurs after insertion of the conserved 1 helix disrupting the dsDNA, thus facilitating the crRNA-DNA hybrid formation. The NT-strand is funnelled towards the RuvC catalytic site, while a long helix of TPID separates the displaced NT-strand and the crRNA-DNA hybrid avoiding DNA re-annealing. The crRNA-DNA hybrid is directed to the stop (STP) domain that splits the hybrid guiding the T-strand towards the RuvC active site. The conserved RuvC insertion of the Cas{Phi} family is extended along the hybrid, interacting with the phosphate backbone of the crRNA. A cluster of hydrophobic residues anchors the RuvC insertion in a cavity of the STP domain. The assembly of the hybrid promotes the shortening of the RuvC insertion, thus pulling the STP towards the RuvC active site to activate catalysis. These findings illustrate why Cas{Phi} unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. Site-directed mutagenesis in key residues support Cas{Phi}3 target DNA and non-specific ssDNA cutting mechanism. Our analysis provides new avenues to redesign the compact CRISPR-Cas{Phi} nucleases for genome editing.

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Carabias del Rey, A., Fuglsang, A., Temperini, P., Pape, T., Sofos, N., Stella, S., Erlendsson, S., Montoya, G.. 2021-06-28. Structure of the mini-RNA-guided endonuclease CRISPR-Cas{Phi}3. https://doi.org/10.1101/2021.06.27.449965

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