A Cas6 functions as an anti-CRISPR of type I-B CRISPR-Cas effector in Thermus thermophilus

Cas6 family ribonucleases can 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, the biological functions of co-occurrence and cross-cleavage of various Cas6 proteins in a multi-CRISPR system host remain largely unexplored. In this study, we biochemically characterized the cross-cleavage of two Cas6 proteins in Thermus thermophilus HB27 and found for the first time 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 the type I Cascade complex. We further demonstrated that Cas6 of type III CRISPR-Cas system could be assembled into I-B Cascade complex, in turn significantly inhibiting the interference activity of type I-B system as an anti-CRISPR. Our findings provide an insight into the functional coupling and regulation mechanisms underlying multiple CRISPR-Cas systems, thereby offering valuable references for designing and controlling type I CRISPR-based gene editing tools precisely.