PRJNA453695
Precursor CRISPR RNA processing and CRISPR adaptation by Cas6-RT-Cas1 protein in Marinomonas mediterranea MMB-1
Prokaryotic CRISPR-Cas systems provide adaptive immunity by integrating portions of invasive nucleic acids (spacers) between genomic CRISPR repeats. Cas6-family proteins help generate spacer-derived tags (crRNAs) that target Cas nucleases to matching invaders. We show that a Marinomonas mediterranea Cas6-reverse transcriptase (RT)-Cas1 fusion combines three biochemical activities (Cas6, RT, and Cas1) that function in both crRNA processing and spacer acquisition from RNA and DNA. We report a 2.85-Å crystal structure of this divergent Cas6, identify its active site by site-directed mutagenesis, and show that the Cas6 domain (but not Cas6 catalytic activity) is required for RT activity and spacer acquisition from RNA. Further, CRISPR repeat RNA binding to Cas6 inhibits the RT, suggesting a mechanism for coordinating these activities. Phylogenetic analysis reveals multiple independent events resulting in stable association of Cas6 with CRISPR-associated RTs, perhaps reflecting a more widespread link between pre-crRNA processing and RNA spacer acquisition.