8Y0D

Crystal structure of SauCas9 in complex with sgRNA and 20nt ssDNA target

Summary for 8Y0D

• Entry DOI: 10.2210/pdb8y0d/pdb
• Descriptor: RNA (73-MER), DNA (5'-D(P*GP*TP*AP*AP*AP*GP*TP*TP*AP*AP*AP*TP*AP*GP*CP*AP*GP*AP*C)-3'), CRISPR-associated endonuclease Cas9, ... (4 entities in total)
• Functional Keywords: crispr, cas12a, crrna, complex, rna binding protein
• Biological source: Staphylococcus aureus; More
• Total number of polymer chains: 3
• Total formula weight: 153893.66

Authors

Chen, Y.,Chen, J.,Liu, L. (deposition date: 2024-01-22, release date: 2025-01-01, Last modification date: 2025-02-19)

Primary citation

Chen, J.,Lin, X.,Xiang, W.,Chen, Y.,Zhao, Y.,Huang, L.,Liu, L.
DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems.
Nucleic Acids Res., 53:-, 2025

PubMed Abstract: Precursor (pre)-CRISPR RNA (crRNA) processing can occur in both the repeat and spacer regions, leading to the removal of specific segments from the repeat and spacer sequences, thereby facilitating crRNA maturation. The processing of pre-crRNA repeat by Cas effector and ribonuclease has been observed in CRISPR-Cas9 and CRISPR-Cas12a systems. However, no evidence of pre-crRNA spacer cleavage by any enzyme has been reported in these systems. In this study, we demonstrate that DNA target binding triggers efficient cleavage of pre-crRNA spacers by type II and V Cas effectors such as Cas12a, Cas12b, Cas12i, Cas12j and Cas9. We show that the pre-crRNA spacer cleavage catalyzed by Cas12a and Cas9 has distinct characteristics. Activation of the cleavage activity in Cas12a is induced by both single-stranded DNA (ssDNA) and double-stranded DNA target binding, whereas only ssDNA target binding triggers cleavage in Cas9 toward the pre-crRNA spacer. We present a series of structures elucidating the underlying mechanisms governing conformational activation in both Cas12a and Cas9. Furthermore, leveraging the trans-cutting activity of the pre-crRNA spacer, we develop a one-step DNA detection method characterized by its simplicity, high sensitivity, and excellent specificity.

PubMed: 39676682
DOI: 10.1093/nar/gkae1241

Experimental method

X-RAY DIFFRACTION (3.92 Å)