Characterizing the transcripts of Leptospira CRISPR I-B array and its processing with endoribonuclease LinCas6

In Leptospira interrogans serovar Copenhageni, the CRISPR-Cas I-B locus possesses a CRISPR array between the two independent cas-operons. Using the reverse transcription-PCR and the in vitro endoribonuclease assay with Cas6 of Leptospira (LinCas6), we account that the CRISPR is transcriptionally active and is conventionally processed. The LinCas6 specifically excises at one site within the synthetic cognate repeat RNA or the repeats of precursor-CRISPR RNA (pre-crRNA) in the sense direction. In contrast, the antisense repeat RNA is cleaved at multiple sites. LinCas6 functions as a single turnover endoribonuclease on its repeat RNA substrate, where substitution of one of predicted active site residues (His38) resulted in reduced activity. This study highlights the comprehensive understanding of the Leptospira CRISPR array transcription and its processing by LinCas6 that is central to RNA-mediated CRISPR-Cas I-B adaptive immunity.