8KAL

Crystal structure of SpyCas9 in complex with sgRNA and 17nt target DNA

Summary for 8KAL

• Entry DOI: 10.2210/pdb8kal/pdb
• Descriptor: RNA (98-MER), CRISPR-associated endonuclease Cas9/Csn1, DNA (25-MER), ... (6 entities in total)
• Functional Keywords: nuclease, rna binding protein/rna/dna, rna binding protein-rna-dna complex
• Biological source: Streptococcus pyogenes serotype M1; More
• Total number of polymer chains: 8
• Total formula weight: 402601.53

Authors

Chen, Y.,Chen, J.,Liu, L. (deposition date: 2023-08-03, release date: 2024-06-05, Last modification date: 2024-06-12)

Primary citation

Chen, J.,Chen, Y.,Huang, L.,Lin, X.,Chen, H.,Xiang, W.,Liu, L.
Trans-nuclease activity of Cas9 activated by DNA or RNA target binding.
Nat.Biotechnol., 2024

PubMed Abstract: Type V and type VI CRISPR-Cas systems have been shown to cleave nonspecific single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA) in trans, but this has not been observed in type II CRISPR-Cas systems using single guide RNA. We show here that the type II CRISPR-Cas9 systems directed by CRISPR RNA and trans-activating CRISPR RNA dual RNAs show RuvC domain-dependent trans-cleavage activity for both ssDNA and ssRNA substrates. Cas9 possesses sequence preferences for trans-cleavage substrates, preferring to cleave T- or C-rich ssDNA substrates. We find that the trans-cleavage activity of Cas9 can be activated by target ssDNA, double-stranded DNA and ssRNA. The crystal structure of Cas9 in complex with guide RNA and target RNA provides a structural basis for the binding of target RNA to activate Cas9. Based on the trans-cleavage activity of Cas9 and nucleic acid amplification technology, we develop the nucleic acid detection platforms DNA-activated Cas9 detection and RNA-activated Cas9 detection, which are capable of detecting DNA and RNA samples with high sensitivity and specificity.

PubMed: 38811761
DOI: 10.1038/s41587-024-02255-7

Experimental method

X-RAY DIFFRACTION (3.16 Å)