Fig. 4: The Anabaena 7120 CRISPR-associated transposase system (AnCAST) and the effects of cvkR deletion and overexpression. | Nature Communications

Fig. 4: The Anabaena 7120 CRISPR-associated transposase system (AnCAST) and the effects of cvkR deletion and overexpression.

From: CvkR is a MerR-type transcriptional repressor of class 2 type V-K CRISPR-associated transposase systems

Fig. 4

a Genes within the 24,963 nt AnCAST include cvkR encoding a transcriptional regulator, cas12k encoding the effector, several cargo and Tn7 genes. Gray triangles indicate hybridization probes used in b–e. RM genes are colored magenta, a toxin-antitoxin module black, other gene functions as in Fig. 1. b to e Northern hybridizations for the cvkR mRNA, the CRISPR array, tracrRNA and cas12k as indicated. Triangles indicate the expected lengths for cvkR, for the CRISPR array (entire length ~250 nt, two repeat-spacer units ~140 nt, partially processed two repeat-spacer unit ~100 nt, single repeat-spacer 70 nt), the tracrRNA and cas12k, a gene of 1.92 kb. Gels and membranes were checked for equal loading by ethidium bromide staining and hybridization to the 120 nt 5S rRNA (in b and d the same membrane was hybridized). The Low Range ssRNA Ladder (NEB) was used as size marker in b and d, the RiboRuler Low and High Range RNA Ladders (Thermo Fisher Scientific) in c and e. Twenty micrograms of total RNA of WT or the respective mutants were separated on 10% PAA-urea (b–d) or 1.5% formaldehyde-agarose gels (e). f Intensities of cas12k mRNA signals normalized to 5S rRNA. Data are presented as mean values ± SD (n = 3 biologically independent samples). The two-sided, equal variance t-test showed distinct expression levels for each strain. g Volcano plot of ΔcvkR and ΔcvkRCom transcriptome analyses. The horizontal line marks the p value cutoff of 0.01, the vertical lines the fold change cutoff of │log2│ ≥ 1. Transcripts above these thresholds showed significant downregulation (blue) or upregulation (red) in ΔcvkR (details in Supplementary Table 3). h The most differentially expressed region in the microarray analysis belongs to the AnCAST system. Individual probes are indicated by horizontal bars, colored in light blue for ΔcvkR and in dark blue for ΔcvkRCom. Analyses shown in b to e were performed twice with consistent results. Source data are provided as a Source Data file.

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