Characterization of type IV-A CRISPR-Cas system

Type IV-A2 CRISPR-Cas systems represent a poorly characterized class of plasmid-encoded CRISPR systems that diverge from other type IV subtypes and were long presumed to lack the Cas8 subunit essential for target recognition. Our recent identification of a conserved, non-canonical Cas8-like protein suggests an alternative mode of complex assembly and interference. Preliminary cryo-EM analyses reveal pronounced structural heterogeneity and conformational flexibility, particularly involving the DinG helicase and Cas6 subunit, which cannot be resolved with existing 200 kV data. We will employ high-throughput cryo-EM data collection at 300 kV to resolve these dynamic states and achieve near-atomic resolution structures. The resulting models will provide the first structural framework for type IV-A2 effector complexes, clarify the roles of DinG and the Cas8-like subunit, and expand current understanding of CRISPR-Cas system diversity and evolution.