Mycobacterium tuberculosis type III‐A CRISPR/Cas system crRNA and its maturation have atypical features

What is it about?

M. tuberculosis, the pathogen that causes TB, is responsible for more deaths than any other bacterial pathogen. Poor understanding of its basic biology hampers efforts to develop more efficacious vaccines and drugs to combat rising levels of multi- and extensive drug resistance. We are interested in the role the CRISPR/Cas system, a sophisticated prokaryotic DNA or RNA-based adaptive immune system for defense against invasive phage and plasmids, plays in M. tuberculosis biology. Variation between strains in its CRISPR locus has been exploited for >20 years in a strain typing system, but the biological function of this Type III-A CRISPR/Cas system requires exploration. Here, we provide the first experimental evidence that the M. tuberculosis CRISPR/Cas system is active in invader defense and document unusual facets of the system. Unusually, its mature crRNA are of one length (～71 nt) and apparently not subject to the 3' end-processing after Cas6 cleavage typical of Type III CRISPR/Cas systems. M. tuberculosis mature crRNA thus resemble Type I system crRNAs, having both a 5' (8 nt) repeat tag and a 3' hairpin loop. Although Cas6 cleavage is generally ion-independent, we found M. tuberculosis Cas6 cleavage to be ion-dependent and accurate cleavage to depend on the repeat RNA 3' hairpin structure and the sequence of its stem base nucleotides. This study not only sheds light on the crRNA recognition mechanism in M. tuberculosis, but also unveils additional diversity among CRISPR/Cas systems and lays a foundation for exploring the development of a Type III-A-based genome editing system.

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