Specialized RNA endonucleases for the maturation of clustered regularly interspaced short palindromic repeat (CRISPR)-derived RNAs (crRNAs) are critical in CRISPR–CRISPR-associated protein (Cas) defence mechanisms. The Cas6 and Cas5d enzymes are the RNA endonucleases in many class 1 CRISPR–Cas systems. In some class 2 systems, maturation and effector functions are combined within a single enzyme or maturation proceeds through the combined actions of RNase III and trans-activating CRISPR RNAs (tracrRNAs). Three separate CRISPR–Cas systems exist in the cyanobacterium Synechocystis sp. PCC 6803. Whereas Cas6-type enzymes act in two of these systems, the third, which is classified as subtype III-B variant (III-Bv), lacks cas6 homologues. Instead, the maturation of crRNAs proceeds through the activity of endoribonuclease E, leaving unusual 13- and 14-nucleotide-long 5′-handles. Overexpression of RNase E leads to overaccumulation and knock-down to the reduced accumulation of crRNAs in vivo, suggesting that RNase E is the limiting factor for CRISPR complex formation. Recognition by RNase E depends on a stem-loop in the CRISPR repeat, whereas base substitutions at the cleavage site trigger the appearance of secondary products, consistent with a two-step recognition and cleavage mechanism. These results suggest the adaptation of an otherwise very conserved housekeeping enzyme to accommodate new substrates and illuminate the impressive plasticity of CRISPR–Cas systems that enables them to function in particular genomic environments.
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We thank O. Alkhnbashi and C. Steglich for helpful comments, U. Ruppert for technical assistance, M. Asayama and E. P. Hudson for the provided plasmids. Financial support from the German Research Foundation programme FOR1680 'Unravelling the Prokaryotic Immune System' (grants HE 2544/8-2 and UR225/1-2) to W.R.H. and H.U. is greatly acknowledged.