Widespread CRISPR repeat-like RNA regulatory elements in CRISPR-Cas systems

Abstract

CRISPR- cas loci typically contain CRISPR arrays with unique spacers separating direct repeats. Spacers along with portions of adjacent repeats are transcribed and processed into CRISPR(cr) RNAs that target complementary sequences (protospacers) in mobile genetic elements, resulting in cleavage of the target DNA or RNA. Additional, standalone repeats in some CRISPR- cas loci produce distinct cr-like RNAs implicated in regulatory or other functions. We developed a computational pipeline to systematically predict crRNA-like elements by scanning for standalone repeat sequences that are conserved in closely related CRISPR- cas loci. Numerous crRNA-like elements were detected in diverse CRISPR-Cas systems, mostly, of type I, but also subtype V-A. Standalone repeats often form mini-arrays containing two repeat-like sequence separated by a spacer that is partially complementary to promoter regions of cas genes, in particular cas8 , or cargo genes located within CRISPR-Cas loci, such as toxins-antitoxins. We show experimentally that a mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide. We also identified mini-arrays in bacteriophages that could abrogate CRISPR immunity by inhibiting effector expression. Thus, recruitment of CRISPR effectors for regulatory functions via spacers with partial complementarity to the target is a common feature of diverse CRISPR-Cas systems.

Figure 1:. Diverse CRISPR repeat-containing RNA molecules in CRISPR-Cas systems

The figure shows previously characterized cr-like RNA molecules encoded in intergenic regions of CRISPR-cas loci (see text for details).

Figure 2:. Computational pipeline for analysis of CRISPR repeat-like sequences.

The pipeline was designed to search for sequences similar to CRISPR repeats outside of CRISPR arrays and predict their possible functions. Cas genes are schematically shown with arrows, and repeat-like sequences are shown with gray boxes.

Figure 3:. CRISPR repeat-like sequences in the CRISPR-cas neighborhoods

The number of BLASTN hits for 4 sets of repeats and mock repeats is plotted against the repeat coverage fraction. Repeat coverage is the length of the sequence detected with BLASTN divided by the query repeat length. A) Repeat-like sequences in intergenic regions. The plot shows the number of BLASTN hits in the CRISPR-cas intergenic regions and intergenic regions of 10 flanking upstream and downstream genes. B) Repeat-like sequences in protein-coding genes. The number of BLASTN hits in the open reading frames of the CRISPR-Cas loci including 10 flanking upstream and downstream genes is shown.

Figure 4:. Putative regulatory mini-arrays in CRISPR-cas loci.

Cas genes are shown as block arrows, repeats are shown as grey boxes annotated with SRU, and spacers and their predicted targets are shown with orange boxes. The locus description includes information on the CRISPR-Cas type, accession for Cas3, nucleotide contig identifier, and start and stop positions of the CRISPR-Cas locus. A) Examples of mini-arrays in I-E, I-F1, I-D, and I-B locus. For comparison, a typical I-E locus that lacks room for an additional RNA gene is shown. B) Detailed organization of a I-E locus containing a mini-array. Green, promoter region; blue box, CRP and H-NS repressor binding sites identifies for type I-E in E.coli (49,50); pink box, LeuO activator sites (51). Sequence regions are color coded as follows: grey, repeat-like sequences; blue, binding sites for H-NS and CRP repressors; orange, spacers and potential targets; green, promoter region. The actual sequence of the mini-array and the predicted duplex between the spacer in its crRNA-like transcript and the cas8 gene promoter is shown underneath the schematic. C) Organization of a I-F1 locus containing a mini-array. Designations are the same as in B. The actual sequence of the mini-array and the predicted duplex between the spacer in its crRNA-like transcript and the cas8 gene promoter is shown underneath the schematic. D) A mini-array in an archaeal genomic locus containing both a I-B and a III-C system.

Figure 5:. The mini-array from a type I-F1 CRISPR-Cas system functions as a regulatory guide RNAs that is not used for interference

A. Gene track alignment of the I-F1 CRISPR-Cas systems from P. aeruginosa strains UCBP-PA14 and YL84. CR is CRISPR mini-array. The predicted target for the mini-array spacer is labeled with a * and the YL84 cascade operon promoter is denoted with an arrow. B. Diagram of a synthetic nanoLuciferase (nLuc) reporter for the YL84 cascade operon. The target sequence is depicted in blue and denoted with a ‘*’, the −35 and −10 sequences are shown as black boxes. C. Relative luminescence assay showing the activity of a nanoluciferase gene placed under the YL84 cascade operon promoter in the presence of variable PA14 I-F1 CRISPR-Cas system components D. Predicted pairing interactions of the YL84 mini-array target with the YL84 mini-array spacer, and a canonical spacer against the same sequence. The PAM is bolded. Every sixth position is expected not to form a base pair, and is depicted in red. Differences in the region 3’ of the spacer sequence are underlined. E. Relative luminescence assay showing the activity of a nanoluciferase gene placed under the YL84 cascade operon promoter in the presence of the PA14 cascade complex and variable crRNA guides. In addition to a no RNA control and the YL84 mini-array RNA, targeting and non-targeting canonical crRNAs were also tested. F. Transformation assay showing plasmid transformation efficiency relative to a non-targeted permissive plasmid. The transformation efficiency of plasmids bearing the YL84 regulatory target or a control target was assayed in strains expressing regulatory, control, or inhibitory crRNA guides as well as PA14 cascade and Cas3. DL is detection limit. ND, not-detected, indicating no colonies were recovered, or the number of colonies was fewer than would allow for quantification.

Figure 6:. Type I-D CRISPR-cas loci containing mini-arrays implicated in the regulation of cargo genes transcription.

A) Cas3 clade with cargo genes located between Cas3 and Cas10. B) Cas3 clade with cargo genes located upstream of the CRISPR-Cas locus. The tree topologies for the two clades of Cas3 tree are shown on the left. Protein-coding genes are shown as block arrows, repeats are shown as gray boxes, spacer and target sequences are shown as red boxes. Cas3 protein accession numbers and the organism names are indicated. Purple circles indicate the presence of a mini-array in the respective locus. For each tree leaf, the locus containing cargo genes is shown.

Figure 7:. Repeat-like sequences in subtype V-A CRISPR-cas loci

Examples of loci containing SRUs from the Francisella, Moraxella, Ruminococcus and Eubacteriales cas12a branches. Cas genes are shown as arrows, SRUs are shown as grey boxes. The locus description includes locus contig, start and stop positions, and the organism name.

Figure 8:. Putative regulatory mini-arrays in phage genomes

Loci containing closely similar mini-arrays in phages and their hosts are shown. A) Clostridium perfringens and Clostridium phage phiCp-D, B) Pseudomonas aeruginosa YL84 and Pseudomonas phage vB_PaS_IME307. Cas genes are shown as arrows, repeats are shown as grey boxes, targeting sequences and targets are shown with orange boxes. The locus description includes locus contig, start and stop positions, and the organism name.

Figure 9:. Prophage encoded regulatory mini-arrays co-occur with cognate CRISPR-Cas systems that lack their own mini-arrays.

Comparison of CRISPR-Cas system-encoded and prophage-encoded mini-arrays found in Acinetobacter baumanii (A-D), and Moraxella osloensis (E-H). A and E, gene track alignments of CRISPR-Cas systems with and without mini-arrays, with genes labeled and regions of high nucleotide identity indicated. CRISPR array sequence is depicted black, while the mini-array target sequence and matching spacers are depicted blue. Mini-arrays are labeled ‘CR,’ and the target sequence is denoted with ‘*’. B and F, prophage loci bearing mini-arrays. Mini-arrays are labeled ‘CR’ with repeats depicted black and spacers depicted blue. ‘DR’ is direct repeats. C and G, alignment of mini-array repeats from host CRISPR-Cas systems and prophages. Matching bases are denoted with a ‘|’. Bases predicted to form stem structures based on RNA base pairing are bolded and italicized. D and H, base pairing between the mini-array crRNA spacer sequences and the putative regulatory target. A putative GG PAM sequence is bolded. Base pairing is depicted with a ‘:’. Every 6th base is depicted in red.