8T6Y

SpRY-Cas9:gRNA complex bound to non-target DNA with 1 bp R-loop

Summary for 8T6Y

• Entry DOI: 10.2210/pdb8t6y/pdb
• EMDB information: 41084
• Descriptor: CRISPR-associated endonuclease Cas9/Csn1, gRNA, TS, ... (4 entities in total)
• Functional Keywords: spry-cas9, crispr, cas9, immune system, r-loop
• Biological source: Streptococcus pyogenes; More
• Total number of polymer chains: 4
• Total formula weight: 193983.59

Authors

Hibshman, G.N.,Bravo, J.P.K.,Taylor, D.W. (deposition date: 2023-06-19, release date: 2024-05-01, Last modification date: 2024-05-15)

Primary citation

Hibshman, G.N.,Bravo, J.P.K.,Hooper, M.M.,Dangerfield, T.L.,Zhang, H.,Finkelstein, I.J.,Johnson, K.A.,Taylor, D.W.
Unraveling the mechanisms of PAMless DNA interrogation by SpRY-Cas9.
Nat Commun, 15:3663-3663, 2024

PubMed Abstract: CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing.

PubMed: 38688943
DOI: 10.1038/s41467-024-47830-3

Experimental method

ELECTRON MICROSCOPY (2.88 Å)