Disparate mechanisms counteract extraneous CRISPR RNA production in type II-C CRISPR-Cas systems

Figure 1.. II-A and II-C CRISPR-Cas systems would both derive an extraneous crRNA from the first repeat adjacent to the newest spacer (II-A) or the oldest spacer (II-C). The newest spacer is acquired through the first repeat in the II-A systems (left) or the last repeat in the II-C systems (right). The CRISPR array is transcribed as a single pre-crRNA (II-A) or as multiple pre-crRNAs due to the repeats containing promoters (II-C). The ecrRNA would be formed at the beginning of the array from the first repeat adjacent to the newest spacer (II-A) or the oldest spacer (II-C).

Figure 2.. Mutation in the extra repeat weakens the interaction energy between extra repeat and tracrRNA. (A) Comparison of the numbers of mutations present in nonextra (left) and extra (right) repeats in CRISPR arrays from type II-C. 364 nonredundant type II-C CRISPR arrays were analysed. Absolute numbers of repeats per category are shown in Table S1. (B) Position of the mutations in the extra and nonextra repeat. 364 type II-C CRISPR arrays were analyzed with a total of 7208 repeats. (C) Interaction between crRNA and tracrRNA of C. jejuni NCTC 11168. RNase III cleavage location is marked with black arrows. (D) Interaction energy between tracrRNA and the extra (left) and the consensus repeat (right). 69 type II-C CRISPR-Cas systems were analyzed (*** P < 1e−5).

Figure 3.. RNA secondary structures found in the intergenic region upstream of the II-C CRISPR arrays can terminate transcription. (A) Up to 180 nt upstream of over 650 the type II-C CRISPR arrays were analyzed for RNA secondary structures and potential RITs were found. The potential RIT in C. jejuni NCTC 11168 directly upstream of the extra repeat was further characterized. (B) The GFP reporter construct design. 84 nucleotides upstream of the first repeat together with 5 nucleotides of the first repeat from C. jejuni NCTC 11168 were cloned into the GFP-expressing plasmid between the promoter and the RBS of the gfp gene. Besides the native sequence, the sequences with the mutations disrupting the stem-loop (SL) or uracil-stretch (Us) of the transcript were tested. (C) Putative structure of the native transcript, including five repeat nucleotides, from C. jejuni cloned into the GFP reporter plasmid. (D) Putative structure of the transcript as in (B) with the mutations disrupting the stem-loop. (E) Putative structure of the transcript as in (B) with the mutations disrupting the uracil-stretch. (F) The GFP fluorescence was measured in E. coli cells transformed with the GFP reporter plasmids…

Figure 4.. Predicted helix formation between the 25 nt ecrRNA spacer and the upstream region. Each dot represents one nucleotide of the ecrRNA and its base-pairing probability with the leader. Left: type II-C with an aggregated P-value of .0026, right: type II-A with an aggregated P-value of .31 as the negative control (eR: extra repeat, ecrRNA: extraneous CRISPR RNA).

Figure 5.. Stem-loop in the ecrRNA spacer of C. jejuni NCTC11168 inhibits targeting of ecrRNA. (A) Putative structure of the extraneous single-guide RNA (esgRNA) from C. jujeni NCTC 11168 predicted with RNAfold. This structure shows the predicted inhibitory hairpin in the spacer, as well as the typical repeat and tracrRNA structures for type II-C. The first nucleotide of the repeat is an A (marked with an arrow) whereas all other repeats of the array start with a G. (B) Putative structure of the extraneous sgRNA with mutations disrupting the stem-loop structure in the spacer (mesgRNA) from C. jujeni NCTC 11168 predicted with RNAfold. (C) Applying the TXTL assay to characterize the targeting activity of the extraneous sgRNA and the impact of the mutations on it. (D) The targeting activity assay was performed in TXTL to compare esgRNA, esgRNA(A→G) (with the repeat starting with a G instead of an A), and mesgRNA. NT sgRNA served as a nontargeting control. The mean (dark-coloured lines) and standard errors (lighter-coloured bars) of two independent measurements are plotted. When the target is recognized on a deGFP-expressing plasmid, the nuclease cleaves the plasmid, which leads to lower…

Figure 6.. Most of the type II-C systems can be assigned to one of the mechanisms potentially preventing the ecrRNA. (A) Systems with mutated repeat are represented by the left circle, with helices in the upstream intergenic region overlapping the ecrRNA spacer or not by the right circle, and with RITs by the lower circle. The numbers account for II-C systems assigned to each subset. 71 out of 260 systems are not assigned to the potential mechanisms. (B) Schematic overview of the different mechanisms and how they prevent the ecrRNA.