9JWK

Cryo-EM structure of FrCas9 in complex with sgRNA and 26-nt TS and 4-nt NTS substrates

Summary for 9JWK

• Entry DOI: 10.2210/pdb9jwk/pdb
• EMDB information: 61853
• Descriptor: CRISPR-associated endonuclease Cas9, sgRNA, DNA (26-mer), ... (4 entities in total)
• Functional Keywords: frcas9, high fidelity, off-target, nuclease protein, complex, hydrolase, hydrolase-rna-dna complex, hydrolase/rna/dna
• Biological source: Faecalibaculum rodentium; More
• Total number of polymer chains: 3
• Total formula weight: 207676.43

Authors

Chen, S.D.,Yang, M.,Liu, S.Q. (deposition date: 2024-10-10, release date: 2025-09-17, Last modification date: 2025-10-29)

Primary citation

Yang, M.,Liu, S.,Chen, G.,Liu, X.,Sun, D.,Zhang, J.,Wang, Y.,Chen, S.,Tian, R.,Hu, Z.
Structural and functional bases of F. rodentium Cas9 provide insights into CRISPR-Cas protein engineering.
Cell Genom, :101039-101039, 2025

PubMed Abstract: The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic promoters. However, the underlying molecular mechanisms remained unexplored. Here, we present cryo-electron microscopy structures of the FrCas9-single guide RNA (sgRNA)-DNA complex in both the R-loop expansion and pre-catalytic states, shedding light on its specialized recognition of the 5'-NRTA-3' protospacer adjacent motif (PAM) and the unusual overwinding of the sgRNA-DNA heteroduplex. Our investigations into the structure and extensive mutational analyses reveal that the phosphate lock loop plays a pivotal role in finely adjusting FrCas9's off-target sensitivity and catalytic efficiency. Remarkably, targeted residue substitutions in the phosphate lock loop and the PAM-distal region were found to synergistically enhance both the editing precision and efficiency of FrCas9. These findings advance our understanding of Cas9's accuracy and potency mechanisms while providing a molecular foundation for the rational design and development of next-generation CRISPR technologies.

PubMed: 41106392
DOI: 10.1016/j.xgen.2025.101039

Experimental method

ELECTRON MICROSCOPY (3.46 Å)