Mechanisms used for cDNA synthesis and site-specific integration of RNAs into DNA genomes by a reverse transcriptase-Cas1 fusion protein

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found that Marinomonas mediterranea RT-Cas1/Cas2 acquires RNA protospacers by adding short 3'-DNA (dN) tails to RNAs protospacers and reverse transcribing these RNAs by multiple mechanisms, including de novo initiation, protein priming, and use of short DNA oligomer primers. Both 3'-dN-RNA/cDNA duplexes and 3'-dN-RNAs in the absence of cDNA synthesis were integrated into CRISPR DNA arrays at rates comparable to similarly configured DNAs. Integration of 3'-dN-RNAs and single-stranded (ss) DNAs was favored at higher proto-spacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNA and ssDNA fragments generated by phage-defense nucleases. Our findings reveal novel mecha-nisms for synthesizing cDNAs of diverse RNAs with no fixed sequence requirements and for site-specifically integrating RNAs into DNA genomes, both with potential biotechnological applications.