Abstract
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR- associated proteins) systems act as microbial adaptive immune systems that protect bacteria against bacteriophages and plasmids. They function by using a complex comprising Cas proteins associated with small CRISPR RNAs (crRNAs) to recognise and destroy invasive nucleic acids. These systems are highly diverse and have been classified into multiple types and subtypes. Type I and type III CRISPR-Cas systems often co-reside in cells and have been shown to interact synergistically to combat invaders. Recently, Cas proteins from a type III-B system were found to use crRNAs from a co-residing type I-F system to provide phage defence in Marinomonas mediterranea MMB-1. In a closely related species, Marinomonas sp. MWYL1, a hybrid of partial type III-B and type I-F systems was discovered that could form an active complex. This system comprises type III-B proteins, a type I-F processing enzyme, and type I-F crRNA. In this study, we showed that the 8 nt sequence at the 5′ end of the crRNA determines formation of the hybrid complex. We tested the ability of the type III-B hybrid proteins to form hybrid complexes with other types of CRISPR-Cas systems by performing individual and high-throughput protein purifications using an assortment of 5′ crRNA sequences. We found that the hybrid type III-B proteins could not form hybrids with other types of CRISPR-Cas systems and are highly specialised for type I-F crRNA. Hybrid systems may endow cells with advantageous properties and since they are likely to have unique functional mechanisms, there is potential to use hybrid systems for novel therapeutic biotechnology and synthetic biology applications.