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Characterization of the self-targeting Type IV CRISPR interference system in Pseudomonas oleovorans

Abstract

Bacterial Type IV CRISPR-Cas systems are thought to rely on multi-subunit ribonucleoprotein complexes to interfere with mobile genetic elements, but the substrate requirements and potential DNA nuclease activities for many systems within this type are uncharacterized. Here we show that the native Pseudomonas oleovorans Type IV-A CRISPR-Cas system targets DNA in a PAM-dependent manner and elicits interference without showing DNA nuclease activity. We found that the first crRNA of P. oleovorans contains a perfect match in the host gene coding for the Type IV pilus biogenesis protein PilN. Deletion of the native Type IV CRISPR array resulted in upregulation of pilN operon transcription in the absence of genome cleavage, indicating that Type IV-A CRISPR-Cas systems can function in host gene regulation. These systems resemble CRISPR interference (CRISPRi) methodology but represent a natural CRISPRi-like system that is found in many Pseudomonas and Klebsiella species and allows for gene silencing using engineered crRNAs.

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Fig. 1: In vivo activity of the P. oleovorans Type IV-A CRISPR-Cas system.
Fig. 2: Self-targeting CRISPR interference of recombinant Type IV-A crRNPs in E. coli.
Fig. 3: Self-targeting Type IV-A CRISPR interference in P. oleovorans cells.
Fig. 4: CRISPRi activity in P. oleovorans using engineered crRNAs.

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Data availability

All data are available in the manuscript or the Extended Data files. Illumina sequence data generated in this study have been deposited in the NCBI Sequence Read Archive database under project ID PRJEB48544. Raw data from single-molecule microscopy analyses are provided at https://doi.org/10.6084/m9.figshare.20359071. Source data are provided with this paper.

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Acknowledgements

We thank S. González Sierra for assistance with flow cytometry; B. Csörgő and J. Bondy-Denomy for providing plasmid pJW31, V. de Lorenzo for providing vector pEMG and A. Davidson for providing plasmid pHERD30T. This work was supported by the DFG-SPP2141 (to X.G. and L.R.), LOEWE Research Cluster Diffusible Signals (to L.R.) and the Max Planck Society (to M.S.-L.).

Author information

Authors and Affiliations

Authors

Contributions

X.G., S.R. and J.W. performed Type IV-A CRISPR-Cas activity assays. J.V.G.-F. analysed the RNA-seq data. M.S-L. performed RT–qPCR analyses. M.S.-L., R.H.-T. and P.L.G. conceived, performed and analysed fluorescence microscopy studies. L.M.I. designed and performed CRISPRi assays in P. oleovorans, and X.G. and P.S. performed CRISPRi assays in E. coli. L.R., X.G. and M.S.-L. conceived the experiments. L.R. wrote the manuscript with support from all other authors.

Corresponding author

Correspondence to Lennart Randau.

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The authors declare no competing interests.

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Nature Microbiology thanks Ryan Jackson, Raymond Staals and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 RNA-seq coverage plots for crRNAs of P. oleovorans.

Illumina RNA-seq analysis revealed 8 nt long 5′-terminal repeat tags for crRNAs of three CRISPR arrays (Type IV: 5′-GUGAGCGG-3′, Type I-E: 5′-AUGAACCG-3′, Type I-F: 5′-CUCAGAAA-3′).

Extended Data Fig. 2 Multiple sequence alignments of conserved Csf1 and DinG sections.

The Clustal X default color scheme is applied47, positions of point mutations investigated in this study are labeled with an asterisk. a. Multiple sequence alignment of Type IV-A Csf1, cysteine residues at position 43, 46, 84 and 87 are conserved. b. Multiple sequence alignment of Type IV-A associated DinG proteins; a variant walker-A motif with consensus sequence TGXGK is identified.

Extended Data Fig. 3 Blue white screening of E. coli colonies with a recombinant Type IV-A CRISPR-Cas system targeting lacZ.

a. Targeting of the genomic lacZ by the recombinant P. oleovorans type IV-A CRISPR-Cas system with point mutation in DinG (K136A) generates only blue colonies. b. Targeting of the genomic lacZ by the recombinant P. oleovorans Type IV-A CRISPR-Cas system generates a mixture of blue and white colonies (left). Individual white colonies (W1-W3) and blue colonies (B1 – B3) were picked for a repeated round of blue white screening. Reversibility of the phenotype was observed.

Extended Data Fig. 4 Recombinant Type IV-A CRISPR targeting of different lacZ regions in E. coli.

a. Overview of targeted protospacers in the genome of E. coli BL21-AI which include the promoter region (1), untranslated region (2), a region of lacZ gene (3) and a downstream region of lacZ (4). A crRNA without lacZ target served as a negative control (C-). Employed spacer sequences are provided in Extended data Table 2. b. Quantification of the observed percentage of blue colonies in blue-white screening of E. coli BL21-AI cells producing Type IV-A crRNPs with indicated target regions. Experiments were performed in triplicates (n = 3 biologically independent colonies). Data are presented as mean values +/−SD. P-values were calculated using unpaired t-test (*p = 0.0132; **p = 0.0032; ****p < 0.0001).

Source data

Extended Data Fig. 5 Single Molecule Tracking of Cas6-mNeonGreen in P. oleovorans.

Representative P. oleovorans cell from the WT and ΔCRISPR strains stained with DAPI for nucleoid visualization and overlying all projection tracks from the SMM analysis. Scale bar 2 µm. b. Standardized cell model containing the projection tracks of a P. oleovorans strain expressing free-diffused mNeonGreen. a-b Experiment was repeated twice (n = 2) using a total of 100 cells per experiment. Scale bar 2 µm. c. Distribution density function of the number of detected fluorophores in all cells. d. Distribution density function of 57329 integrated spot intensities. In the best estimation, there are two populations of average integrated intensity I1 = 29341 and I2 = 27901 u.a., with a proportion of 57%/43%. Therefore, the estimation of the number of fluorophores after accounting for crRNP complex formation and simulation corrections is 26 per cell.

Extended Data Fig. 6 Gene silencing assay for trpE in P. oleovorans plated on minimal medium agar.

The control strain is carrying an empty pHERD30T plasmid instead of pHERD30T coding for the crRNA targeting trpE. Experiments were performed in triplicates (n = 3 biologically independent samples) and four dilutions (10−2–10−5) were plated, respectively. Statistical analysis was performed using an unpaired t-test. Data are presented as mean values +/−SD with a p = 0.0069 (**).

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Identification of PAM sequences for perfect protospacer matches of indicated P. oleovorans CRISPR array spacers.

Supplementary Table 2

List of spacers used for Type IV-A CRISPR-Cas targeting.

Supplementary Table 3

List of differentially expressed genes with significant adjusted P value identified from the RNA-seq data comparing P. oleovorans WT and ΔCRISPR array strains.

Supplementary Table 4

Plasmids used for genetic manipulation of P. oleovorans.

Supplementary Table 5

Primers used for RT–qPCR.

Source data

Source Data Fig.1d

Source data of the flow cytometry experiment (Fig. 1d).

Source Data Fig. 1d–h

Raw data used for construction and statistical analysis of Fig. 1d–h.

Source Data Fig. 2a

LB agar plates with X-Gal containing the colonies of the lacZ target experiment. These plates were used for constructing Fig. 2a.

Source Data Fig. 2b

Raw data used for construction and statistical analysis of Fig. 2b.

Source Data Fig. 3

Raw data used for construction and statistical analysis of Fig. 3f.

Source Data Fig. 4

Raw data used for construction and statistical analysis of Fig. 4a,b.

Source Data Extended Data Fig. 4

Raw data used for construction and statistical analysis of Extended Data Fig. 4b.

Source Data Extended Data Fig. 6

Raw data used for construction and statistical analysis of Extended Data Fig. 6.

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Guo, X., Sanchez-Londono, M., Gomes-Filho, J.V. et al. Characterization of the self-targeting Type IV CRISPR interference system in Pseudomonas oleovorans. Nat Microbiol 7, 1870–1878 (2022). https://doi.org/10.1038/s41564-022-01229-2

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