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Abstract:

CRISPR-Cas adaptive immunity loci typically contain CRISPR arrays that harbor unique spacers between direct repeats. Spacers along with portions of adjacent repeats are transcribed into CRISPR(cr) RNAs that mostly target sequences complementary to spacers (protospacers) in mobile genetic elements, such as viruses or plasmids, in most cases, resulting in cleavage or degradation of the target DNA or RNA. Additionally, standalone repeats were detected in the vicinity of some CRISPR loci, for which adjacent sequences can serve as spacers so that a distinct cr-like RNA is expressed that can be employed for regulatory or other functions. The best-characterized case of standalone repeat neofunctionalization is the tracrRNA of type II and some type V CRISPR systems. In other CRISPR systems, standalone repeats with spacer-like sequences regulate toxin production or bacterial virulence. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR-cas loci. We predict numerous novel crRNA-like elements in diverse CRISPR-Cas systems, mostly, of type I, but also of subtype V-A. Many of the standalone repeats form mini-arrays that contain two repeat-like sequence, the distal one typically degenerate, separated by a spacer-like sequence. The spacer-like sequences often target promoter regions of cas genes, in particular cas8, or cargo genes located within CRISPR-Cas loci, particularly, toxin-antitoxin modules. This targeting specificity strongly suggests that crRNA-like transcripts of the mini-arrays perform regulatory functions in particular, repression of cas effector gene transcription, mitigating the cost of CRISPR-Cas maintenance. Other standalone repeat-like sequences are single repeat units potentially transcribed into short RNAs that could bind and inhibit CRISPR effectors, and facilitate recombination between CRISPR-cas loci.