A study published in Nature Communications and conducted by researchers from the Institute of Physics of the Chinese Academy of Sciences revealed new details about the CRISPR-Cas5-HNH/Cascade complex, a variant of the type I-E CRISPR-Cas system, providing new insights into its DNA recognition and cleavage mechanisms.
The CRISPR-Cas5-HNH/Cascade complex, which serves as an immune defense system in prokaryotes, has long been studied for its ability to protect bacteria from invading genetic materials. However, recent research uncovers a novel mechanism in this complex that differs significantly from the well-known type II CRISPR systems like Cas9.
Researchers utilized cryo-electron microscopy to capture the structures of the Cas5-HNH/Cascade complex in both DNA-bound and unbound states. They revealed striking conformational changes. The complex adopts a more compact structure when bound to double-stranded DNA (dsDNA), with the target DNA strand making a pronounced U-turn and interacting with the HNH nuclease domain. The target strand is cleaved first, followed by the non-target strand, a departure from the cleavage order seen in other CRISPR systems like Cas12a.
The Cas5-HNH domain itself was found to play a crucial role in the nuclease activity of the Cas5-HNH/Cascade complex. Researchers demonstrated that mutations in key residues of the HNH domain, particularly histidine and aspartate, can completely abolish cleavage activity. This suggested that the HNH domain is not only essential for target DNA recognition but also for the specific cleavage of DNA, further differentiating the mechanism from other systems.
Moreover, researchers observed that the architecture of the Cas5-HNH/Cascade complex is notably different from canonical type I-E systems. For example, the HNH domain was fused to the C-terminus of Cas5, replacing the need for Cas3, a protein commonly involved in DNA degradation in other systems. This structural innovation pointed to the complex's ability to cleave DNA independently of the traditional components seen in similar CRISPR systems.
By elucidating the role of the HNH nuclease domain and revealing the structural changes that occur upon DNA binding, this work not only deepens our understanding of CRISPR-Cas5-HNH but also paves the way for refining CRISPR technologies for therapeutic applications.
