Adaptive immunity of type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins

Cas13-containing type VI CRISPR-Cas systems specifically target RNA; however, the mechanism of spacer acquisition remains unclear. Using computational analyses, we have previously reported the association of RT-Cas1 fusion proteins with type VI-A systems. Here, we found that RT-Cas1 fusion proteins were also recruited by type VI-B systems in bacteria from gut metagenomes, constituting a VI-B1 variant system that comprises a CorA-encoding locus in addition to the CRISPR array and adaptation RT-Cas1/Cas2 module. We found that type VI RT-CRISPR systems are functional in spacer acquisition, CRISPR array processing, and interference activity, demonstrating that their adaptive immunity can function independently of other in-trans systems. We provide evidence that in these systems, the associated RT enables spacer acquisition from the RNA molecules. Additionally, we found that the encoded CorA in type VI-B1 RT-associated systems can transport divalent metal ions and downregulate Cas13b-mediated RNA interference. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, which may enable the integration of RNA-derived spacers into the CRISPR array as a mechanism against RNA-based invaders in specific environments.