An expanded CRISPR–Cas9-assisted recombineering toolkit for engineering genetically intractablePseudomonas aeruginosaisolates

Debbie Pankratz, Nicolas Oswaldo Gomez, Agnes Nielsen, Ayten Mustafayeva, Melisa Gür, Fabián Arce-Rodriguez, Pablo Ivan Nikel, Susanne Häussler, Alejandro Arce-Rodriguez

Published: 2023-10-04 DOI: 10.1038/s41596-023-00882-z

CRISPR-Cas9

Pseudomonas aeruginosa

recombineering

genetic engineering

Ssr recombinase

AI 解读

Supplementary information

Supplementary Table 1

Oligonucleotides used in this study.

Generation of mouse–human chimeric embryos

Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase footprinting

Generation of CRISPR–Cas9-mediated genetic knockout human intestinal tissue–derived enteroid lines by lentivirus transduction and single-cell cloning

A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues

Decoding personal biotic and abiotic airborne exposome

A clinically relevant model of focal embolic cerebral ischemia by thrombus and thrombolysis in rhesus monkeys

Bioengineering human intestinal mucosal grafts using patient-derived organoids, fibroblasts and scaffolds

Biomimetic tail-to-head terpene cyclizations using the resorcin[4]arene capsule catalyst

Neuroscience robotics for controlled induction and real-time assessment of hallucinations

Scanorama: integrating large and diverse single-cell transcriptomic datasets

Single-cell RNA sequencing (scRNA-seq)（单细胞RNA测序）

Data integration（数据集成）

Heterogeneity（异质性）

查看全部

Sections

Figures

References

Supplementary information

Silby, M. W., Winstanley, C., Godfrey, S. A., Levy, S. B. & Jackson, R. W. Pseudomonas genomes: diverse and adaptable. FEMS Microbiol. Rev. 35, 652–680 (2011).
Tacconelli, E. et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect. Dis. 18, 318–327 (2018).
Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399, 629–655 (2022).
Lee, D. G. et al. Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial. Genome Biol. 7, R90 (2006).
Klockgether, J., Cramer, N., Wiehlmann, L., Davenport, C. F. & Tummler, B. Pseudomonas aeruginosa genomic structure and diversity. Front. Microbiol. 2, 150 (2011).
Freschi, L. et al. The Pseudomonas aeruginosa pan-genome provides new insights on its population structure, horizontal gene transfer, and pathogenicity. Genome Biol. Evol. 11, 109–120 (2019).
Ciofu, O., Riis, B., Pressler, T., Poulsen, H. E. & Hoiby, N. Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation. Antimicrob. Agents Chemother. 49, 2276–2282 (2005).
Rossi, E. et al. Pseudomonas aeruginosa adaptation and evolution in patients with cystic fibrosis. Nat. Rev. Microbiol. 19, 331–342 (2021).
Culyba, M. J. & Van Tyne, D. Bacterial evolution during human infection: adapt and live or adapt and die. PLoS Pathog. 17, e1009872 (2021).
Schweizer, H. P. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol. Microbiol. 6, 1195–1204 (1992).
Hoang, T. T., Karkhoff-Schweizer, R. R., Kutchma, A. J. & Schweizer, H. P. A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212, 77–86 (1998).
Choi, K. H. & Schweizer, H. P. An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants. BMC Microbiol. 5, 30 (2005).
Shanks, R. M., Caiazza, N. C., Hinsa, S. M., Toutain, C. M. & O’Toole, G. A. Saccharomyces cerevisiae-based molecular tool kit for manipulation of genes from gram-negative bacteria. Appl. Environ. Microbiol. 72, 5027–5036 (2006).
Martinez-Garcia, E. & de Lorenzo, V. Engineering multiple genomic deletions in Gram-negative bacteria: analysis of the multi-resistant antibiotic profile of Pseudomonas putida KT2440. Environ. Microbiol. 13, 2702–2716 (2011).
Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat. Protoc. 10, 1820–1841 (2015).
Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
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).
Aparicio, T., Jensen, S. I., Nielsen, A. T., de Lorenzo, V. & Martinez-Garcia, E. The Ssr protein (T1E_1405) from Pseudomonas putida DOT-T1E enables oligonucleotide-based recombineering in platform strain P. putida EM42. Biotechnol. J. 11, 1309–1319 (2016).
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. Usa. 97, 6640–6645 (2000).
Lesic, B. & Rahme, L. G. Use of the lambda Red recombinase system to rapidly generate mutants in Pseudomonas aeruginosa. BMC Mol. Biol. 9, 20 (2008).
Aparicio, T., de Lorenzo, V. & Martinez-Garcia, E. CRISPR/Cas9-based counterselection boosts recombineering efficiency in Pseudomonas putida. Biotechnol. J. 13, e1700161 (2018).
Aparicio, T., de Lorenzo, V. & Martinez-Garcia, E. CRISPR/Cas9-enhanced ssDNA recombineering for Pseudomonas putida. Microb. Biotechnol. 12, 1076–1089 (2019).
Jeske, A., Arce-Rodriguez, A., Thoming, J. G., Tomasch, J. & Haussler, S. Evolution of biofilm-adapted gene expression profiles in lasR-deficient clinical Pseudomonas aeruginosa isolates. NPJ Biofilms Microbiomes 8, 6 (2022).
Calero, P. & Nikel, P. I. Chasing bacterial chassis for metabolic engineering: a perspective review from classical to non-traditional microorganisms. Microb. Biotechnol. 12, 98–124 (2019).
Xin, X. F., Kvitko, B. & He, S. Y. Pseudomonas syringae: what it takes to be a pathogen. Nat. Rev. Microbiol. 16, 316–328 (2018).
Chen, W. et al. CRISPR/Cas9-based genome editing in Pseudomonas aeruginosa and cytidine deaminase-mediated base editing in Pseudomonas species. iScience 6, 222–231 (2018).
Ricaurte, D. E. et al. A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities. Microb. Biotechnol. 11, 176–188 (2018).
Volke, D. C., Martino, R. A., Kozaeva, E., Smania, A. M. & Nikel, P. I. Modular (de)construction of complex bacterial phenotypes by CRISPR/nCas9-assisted, multiplex cytidine base-editing. Nat. Commun. 13, 3026 (2022).
Konstantakos, V., Nentidis, A., Krithara, A. & Paliouras, G. CRISPR–Cas9 gRNA efficiency prediction: an overview of predictive tools and the role of deep learning. Nucleic Acids Res. 50, 3616–3637 (2022).
Blin, K., Pedersen, L. E., Weber, T. & Lee, S. Y. CRISPy-web: an online resource to design sgRNAs for CRISPR applications. Synth. Syst. Biotechnol. 1, 118–121 (2016).
Concordet, J. P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018).
Aparicio, T. et al. Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene. Environ. Microbiol. 22, 45–58 (2020).
Silva-Rocha, R. et al. The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res. 41, D666–D675 (2013).
Martinez-Garcia, E. et al. SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes. Nucleic Acids Res. 51, D1558–D1567 (2023).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. Improving CRISPR–Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).
Ran, F. A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).
Wang, T., Wei, J. J., Sabatini, D. M. & Lander, E. S. Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014).
Lin, Y. et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 (2014).
Xu, H. et al. Sequence determinants of improved CRISPR sgRNA design. Genome Res. 25, 1147–1157 (2015).
Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR–Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).
Liang, G., Zhang, H., Lou, D. & Yu, D. Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing. Sci. Rep. 6, 21451 (2016).
Ellis, H. M., Yu, D., DiTizio, T. & Court, D. L. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl Acad. Sci. USA 98, 6742–6746 (2001).
Shen, H., Han, F., Lin, Y. & Yu, W. A high efficient electroporation of Pseudomonas sp. QDA pretreated with alginate lyase. Enzym. Microb. Technol. 39, 677–682 (2006).
Hanahan, D. & Meselson, M. in Methods in Enzymology (eds. Wu, R. et al.) Vol. 100, 333–342 (Academic Press, 1983).
Rahme, L. G. et al. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268, 1899–1902 (1995).
Holloway, B. W. Genetic recombination in Pseudomonas aeruginosa. Microbiology 13, 572–581 (1955).
Hornischer, K. et al. BACTOME—a reference database to explore the sequence- and gene expression-variation landscape of Pseudomonas aeruginosa clinical isolates. Nucleic Acids Res. 47, D716–D720 (2019).
Boyer, H. W. & Roulland-Dussoix, D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41, 459–472 (1969).
Keen, N. T., Tamaki, S., Kobayashi, D. & Trollinger, D. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70, 191–197 (1988).
Kanehisa, M. & Goto, S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 28, 27–30 (2000).
Winsor, G. L. et al. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database. Nucleic Acids Res. 44, D646–D653 (2016).
Benson, D. A. et al. GenBank. Nucleic Acids Res 41, D36–D42 (2013).
Poulsen, B. E. et al. Defining the core essential genome of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 116, 10072–10080 (2019).
Liberati, N. T. et al. An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants. Proc. Natl Acad. Sci. USA 103, 2833–2838 (2006).
Jacobs, M. A. et al. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 100, 14339–14344 (2003).
Hofacker, I. L. Vienna RNA secondary structure server. Nucleic Acids Res. 31, 3429–3431 (2003).
Kessler, B., de Lorenzo, V. & Timmis, K. N. A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy. Mol. Gen. Genet. 233, 293–301 (1992).
Simon, R., Priefer, U. & Pühler, A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat. Biotechnol. 1, 784–791 (1983).
Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd edn (Cold Spring Harbor Laboratory, 1989).
Roland, K., Curtiss, R. 3rd & Sizemore, D. Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens. Avian Dis. 43, 429–441 (1999).
Choi, K. H., Kumar, A. & Schweizer, H. P. A 10-min method for preparation of highly electrocompetent Pseudomonas aeruginosa cells: application for DNA fragment transfer between chromosomes and plasmid transformation. J. Microbiol. Methods 64, 391–397 (2006).

An expanded CRISPR–Cas9-assisted recombineering toolkit for engineering genetically intractablePseudomonas aeruginosaisolates

Supplementary information

Supplementary Table 1

推荐阅读