Cas5 dimer structure offers clues for CRISPR system engineering

While Cas5 is not a direct genome editor, it serves as a modular, engineerable component in multiprotein CRISPR systems that offer advantages over Cas9-based tools – including enhanced specificity through two-step target recognition and the capacity for large-scale DNA deletions relevant to antimicrobial and antiviral applications.

The research team demonstrated that MboCas5 forms stable dimers via salt bridges between the R72 and D167 residues; however, when they disrupted dimerisation with the R72W mutation, the resulting monomer retained full crRNA processing activity. This functional equivalence has important practical implications – researchers working with isolated Cas5 for in vitro RNA processing need not maintain specific oligomeric states. However, structural modelling revealed that dimeric Cas5 creates steric clashes with Cas8 in assembled Cascade complexes, explaining why only monomers incorporate into functional surveillance machinery.

The team also identified a flexible loop region undergoing substantial conformational rearrangement during Cascade assembly. For researchers engineering type I-C systems, modulating the R72-D167 interface could control Cas5 availability for complex formation, whilst the flexible loop represents a potential target for allosteric regulation or stability optimisation.

The study was led by Yong Jun Kang, Hyun Ji Ha and Hyun Ho Park at Chung-Ang University, Seoul, Republic of Korea. It was published in Scientific Reports on 15 December 2025.