Differential processing of CRISPR RNA by LinCas5c and LinCas6 of Leptospira

Leptospira interrogans serovar Copenhageni's genome harbors two CRISPR-Cas systems belonging to subtypes IB and IC. However, in L. interrogans, the subtype IC locus lacks an array component essential for assembling an interference complex. Thus, the reason for sustaining the expense of a cluster of cas genes (IC) is obscure. Type IC (previously Dvulg) is the only CRISPR subtype that engages Cas5c, a Cas5 variant, to process precursor CRISPR-RNA (pre-crRNA). In this study, thus, the recombinant Cas5c (rLinCas5c) of L. interrogans and its mutant variants were cloned, expressed, and purified. The purified rLinCas5c is illustrated as metal-independent, sequence, and size-specific cleavage on repeat RNA and pre-crRNA of subtype IB or orphan CRISPR array. However, the Cas6-bound mature crRNA of subtype IB fends off from the rLinCas5c activity. In addition, rLinCas5c holds metal and size-dependent DNase activity. The bioinformatics analysis of LinCas5c inferred that it belongs to the subgroup Cas5c-B. Substitution of Phe141 with a more conserved His residue and deletion of unique (β1′-β2′) insertions usher a gain of rLinCas5c activity over nucleic acid. Overall, our results uncover the functional diversity of Cas5c ribonucleases and infer an incognito auxiliary role in the absence of a cognate CRISPR array. •Recombinant LinCas5c demonstrates non-canonical cleavage activity on repeat RNA substrate.•Standalone rLinCas5c is not capable of generating mature CRISPR RNA from precursor CRISPR RNA.•LinCas6 in subtype IB may protect crRNA from co-existing endoribonucleases such as LinCas5c in Leptospira.•LinCas5c is a member of the Cas5c-B subgroup.•Recombinant LinCas5c exhibit metal-dependent DNase activity on larger DNA substrate (≥ 2.5 kb); however, found to be inert towards smaller DNA substrate (36 bp).•LinCas5c contains Phe (141) in its catalytic triad instead of His as that of its orthologs, and the substitution of Phe with His (141) leads to a gain in nuclease activity.•LinCas5c exclusively contains β-sheet insertion at its C-terminal region, and its deleted mutant leads to a gain in nuclease activity.