Abstract
Bacteria have adaptive defence mechanisms that protect them from phage infection. One defence mechanism is called CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins), which uses ribonucleoprotein surveillance complexes to degrade invading viral nucleic acids. CRISPR-Cas systems can be divided into many types and subtypes that vary in composition and mode of action. Type I and III CRISPR-Cas systems utilise multiprotein complexes, which differ in structure and cleave either dsDNA or ssRNA and ssDNA, respectively. The type I-D system is a chimera of type I and III systems, resulting in the composition and mechanism of the surveillance complex being unclear. One protein common in type I and III surveillance complexes is the small subunit. However, many type I systems, such as type I-D, do not possess a small subunit gene and it was predicted that the C-terminus of the large subunit was extended and functionally substituted for the small subunit. In the type I-D system, we demonstrated that small subunits were expressed from an alternative translational start site within the large subunit gene. These small subunits formed the minor filament of the complex and played an important role in its interaction with dsDNA. We have also shown that the internal translation of small subunit genes may be conserved across not only type I-D systems but also type I-B and I-C systems, which together comprise of ~23% of all known CRISPR-Cas systems. Furthermore, we showed that the type I-D complex could bind dsDNA, ssDNA, and ssRNA, and it uses independent mechanisms to detect the double-stranded from the single-stranded targets. We solved the structures of the type I-D complex bound to dsDNA and ssRNA, which revealed that the overall architecture was more comparable to type III systems than type I. Overall, the chimeric nature of the type I-D system provides a potential novel biotechnological tool, and the further understanding of this system allows us to visualise a step between the evolution of type I from type III CRISPR-Cas systems.