Structural Basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex

Type I-E CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) system is the most widely investigated RNA-guided adaptive immune system in prokaryotes against the foreign genetic elements. Unlike the previously characterized Cas3 nuclease in the typical Type I-E system that exhibits uncontrolled progressive DNA cleavage, a recently discovered HNH domain linked to the Cas5 subunit functions as the nuclease for precise DNA cleavage, highlighting the potential of this new Type I-E variant system as a precise genome editing tool. Here, we present five near- atomic cryo-EM (cryo-electron microscopy) structures of Candidatus Cloacimonetes bacterium Cas5-HNH/Cascade complex either bound or unbound to DNA. We revealed that the HNH domain extensively interacts with the adjacent subunits, including Cas6, Cas8 and Cas11. The related mutations of the crucial identified interactions can significantly weaken the performance of the enzyme. Upon the binding of DNA, the Cas-HNH/Cascade complex adopts a more compacted conformation with the subunits moving towards the center of the nuclease, thus activating the nuclease. We further identified four conserved cysteines that form a zinc-finger structure in the HNH domain and the mutations of these four cysteines can totally abolish the enzyme activity. Interestingly, we also discovered that the divalent ions such as zinc, cobalt and nickel down-regulate the enzyme performance by decreasing the stability of the Cascade complex. Together, our findings provide first structural insights into the assembly and activation of the Cas5-HNH/Cascade complex, opening new avenues for engineering this system for precise genome editing.