An Active Immune Defence with a Minimal CRISPR (clustered regularly interspaced short palindromic repeats) RNA and Without the Cas6 Protein [Microbiology]

December 15th, 2014 by Maier, L.-K., Stachler, A.-E., Saunders, S. J., Backofen, R., Marchfelder, A.

The prokaryotic immune system CRISPR-Cas1 is a defence system that protects prokaryotes against foreign DNA. The short CRISPR RNAs (crRNAs) are central components of this immune system. In CRISPR-Cas systems type I and III crRNAs are generated by the endonuclease Cas6. We developed a Cas6b2-independent crRNA maturation pathway for the Haloferax type I-B system in vivo, that expresses a functional crRNA that we termed independently generated crRNA (icrRNA). The icrRNA is effective in triggering degradation of an invader plasmid carrying the matching protospacer sequence. The Cas6b-independent maturation of the icrRNA allowed mutation of the repeat sequence without interfering with signals important for Cas6b processing. We generated 23 variants of the icrRNA and analysed them for activity in the interference reaction. icrRNAs with deletions or mutations of the 3′ handle are still active in triggering a interference reaction. The complete 3′ handle could be removed without loss of activity. However manipulations of the 5′ handle mostly led to loss of interference activity. Furthermore we could show that in the presence of an icrRNA a strain without Cas6b (∆cas6b) is still active in interference.
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