8SQH
SpRY-Cas9:gRNA complex targeting TTC PAM DNA
Summary for 8SQH
| Entry DOI | 10.2210/pdb8sqh/pdb |
| Related | 8SPQ |
| EMDB information | 40681 40705 |
| Descriptor | CRISPR-associated endonuclease Cas9/Csn1, gRNA, TS, ... (6 entities in total) |
| Functional Keywords | spry-cas9, crispr, cas9, immune system, immune system-hydrolase complex, immune system/hydrolase |
| Biological source | Streptococcus pyogenes More |
| Total number of polymer chains | 5 |
| Total formula weight | 217679.80 |
| Authors | Hibshman, G.N.,Bravo, J.P.K.,Taylor, D.W. (deposition date: 2023-05-04, 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 Cited by 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: 38688943DOI: 10.1038/s41467-024-47830-3 PDB entries with the same primary citation |
| Experimental method | ELECTRON MICROSCOPY (3.69 Å) |
Structure validation
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