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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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.
References
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
Marraffini, L. A. & Sontheimer, E. J. Self versus non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568–571 (2010).
Bolotin, A., Quinquis, B., Sorokin, A. & Ehrlich, S. D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151, 2551–2561 (2005).
Mojica, F. J. M., Diez-Villasenor, C., Garcia-Martinez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).
Nunez, J. K. et al. Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity. Nat. Struct. Mol. Biol. 21, 528–534 (2014).
Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).
Hale, C. R. et al. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex. Cell 139, 945–956 (2009).
Marraffini, L. A. CRISPR-Cas immunity in prokaryotes. Nature 526, 55–61 (2015).
Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J. & Soria, E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J. Mol. Evol. 60, 174–182 (2005).
Pourcel, C., Salvignol, G. & Vergnaud, G. CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653–663 (2005).
Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR-Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).
Makarova, K. S. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).
Pinilla-Redondo, R. et al. Type IV CRISPR-Cas systems are highly diverse and involved in competition between plasmids. Nucleic Acids Res. 48, 2000–2012 (2020).
Kamruzzaman, M. & Iredell, J. R. CRISPR-Cas system in antibiotic resistance plasmids in Klebsiella pneumoniae. Front. Microbiol. 10, 2934 (2019).
Makarova, K. S. et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020).
Özcan, A. et al. Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum. Nat. Microbiol. 4, 89–96 (2019).
Staals, R. H. J. et al. RNA targeting by the type III-A CRISPR-Cas Csm complex of Thermus thermophilus. Mol. Cell 56, 518–530 (2014).
Leenay, R. T. et al. Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol. Cell 62, 137–147 (2016).
Crowley, V. M. et al. A type IV-A CRISPR-Cas system in Pseudomonas aeruginosa mediates RNA-guided plasmid interference in vivo. CRISPR J. 2, 434–440 (2019).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Jia, N. et al. Type III-A CRISPR-Cas Csm complexes: assembly, periodic RNA cleavage, DNase activity regulation, and autoimmunity. Mol. Cell 73, 264–277.e5 (2019).
Westra, E. R. et al. Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition. PLoS Genet. 9, e1003742 (2013).
Taylor, H. N. et al. Positioning diverse type IV structures and functions within Class 1 CRISPR-Cas systems. Front. Microbiol. 12, 671522 (2021).
Cass, S. D. et al. The role of Cas8 in type I CRISPR interference. Biosci. Rep. 35, e00197 (2015).
Li, Z., Zhang, H., Xiao, R. & Chang, L. Cryo-EM structure of a type I-F CRISPR RNA guided surveillance complex bound to transposition protein TniQ. Cell Res. 30, 179–181 (2020).
Makarova, K. S., Aravind, L., Wolf, Y. I. & Koonin, E. V. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems. Biol. Direct 6, 38 (2011).
Makarova, K. S. et al. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9, 467–477 (2011).
Csorgo, B. et al. A compact Cascade-Cas3 system for targeted genome engineering. Nat. Methods 17, 1183–1190 (2020).
Oviedo-Bocanegra, L. M., Hinrichs, R., Rotter, D. A. O., Dersch, S. & Graumann, P. L. Single molecule/particle tracking analysis program SMTracker 2.0 reveals different dynamics of proteins within the RNA degradosome complex in Bacillus subtilis. Nucleic Acids Res. 49, e112 (2021).
van Schaik Erin, J. et al. DNA binding: a novel function of Pseudomonas aeruginosa type IV pili. J. Bacteriol. 187, 1455–1464 (2005).
Mulepati, S. & Bailey, S. Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 3 (Cas3). J. Biol. Chem. 286, 31896–31903 (2011).
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Thrash, A., Arick, M. 2nd & Peterson, D. G. Quack: a quality assurance tool for high throughput sequence data. Anal. Biochem. 548, 38–43 (2018).
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Wickham, H. ggplot2: Elegant Graphics for Data Analysis (Springer-Verlag, 2016).
Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).
Wirth, N. T., Kozaeva, E. & Nikel, P. I. Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection. Microb. Biotechnol. 13, 233–249 (2020).
Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat. Protoc. 10, 1820–1841 (2015).
Geissmann, Q. OpenCFU, a new free and open-source software to count cell colonies and other circular objects. PLoS ONE 8, e54072 (2013).
Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001).
Hernández-Tamayo, R., Schmitz, H. & Graumann, P. L. Single-molecule dynamics at a bacterial replication fork after nutritional downshift or chemically induced block in replication. mSphere 6, e00948–20 (2021).
Paintdakhi, A. et al. Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis. Mol. Microbiol. 99, 767–777 (2016).
Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702 (2008).
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882 (1997).
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.).
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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.
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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).
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 (**).
Supplementary information
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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DOI: https://doi.org/10.1038/s41564-022-01229-2
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