Structural constraints and enzymatic promiscuity in the Cas6-dependent generation of crRNAs

A hallmark of defense mechanisms based on clustered regularly interspaced short palindromic repeats (CRISPR) and associated sequences (Cas) are the crRNAs that guide these complexes in the destruction of invading DNA or RNA. Three separate CRISPR-Cas systems exist in the cyanobacterium Synechocystis sp. PCC 6803. Based on genetic and transcriptomic evidence, two associated endoribonucleases, Cas6-1 and Cas6-2a, were postulated to be involved in crRNA maturation from CRISPR1 or CRISPR2, respectively. Here, we report a promiscuity of both enzymes to process in vitro not only their cognate transcripts, but also the respective non-cognate precursors, whereas they are specific in vivo Moreover, while most of the repeats serving as substrates were cleaved in vitro, some were not. RNA structure predictions suggested that the context sequence surrounding a repeat can interfere with its stable folding. Indeed, structure accuracy calculations of the hairpin motifs within the repeat sequences explained the majority of analyzed cleavage reactions, making this a good measure for predicting successful cleavage events. We conclude that the cleavage of CRISPR1 and CRISPR2 repeat instances requires a stable formation of the characteristic hairpin motif, which is similar between the two types of repeats. The influence of surrounding sequences might partially explain variations in crRNA abundances and should be considered when designing artificial CRISPR arrays.