A type III-associated Cas6 functions as a negative regulator of type I-B CRISPR-Cas system in Thermus thermophilus

CRISPR-Cas systems are small RNA-guided immune systems in prokaryotes. CRISPR RNA (crRNA) provides sequence specificity and programmability, guiding the effector complex to cleave target nucleic acids. Cas6 family ribonucleases can cleave precursor crRNA to generate functional crRNAs in most type I and type III CRISPR-Cas systems. Most existing studies of Cas6 functions are mainly focused on nuclease activity in vitro and Cas6-processed product characterization in vivo. However, in hosts harboring multiple CRISPR systems, the biological functions of the co-occurrence of various Cas6 proteins and their cross-cleavage activity toward different types of crRNAs remain largely unexplored. In this study, we biochemically characterized the cross-cleavage activity of two Cas6 proteins in Thermus thermophilus HB27 and first found that Cas6 could anchor the mature crRNA and interact with Cas5 subunit of type I-B system, revealing the functions of Cas6 to mediate the assembly of type I Cascade complex. We further demonstrated that the type III-associated Cas6 protein could act as a negative regulator by competing with the I-B Cas6 protein during the assembly of type I-B Cascade complex, significantly suppressing the interference activity of type I-B system. Our findings provide an insight into the functional coupling and regulation mechanisms underlying multiple CRISPR-Cas systems.