Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Precise genome engineering in Pseudomonas using phage-encoded homologous recombination and the Cascade–Cas3 system

Nature Protocols volume 18, pages 2642–2670 (2023)Cite this article

Subjects

Abstract

A lack of generic and effective genetic manipulation methods for Pseudomonas has restricted fundamental research and utilization of this genus for biotechnology applications. Phage-encoded homologous recombination (PEHR) is an efficient tool for bacterial genome engineering. This PEHR system is based on a lambda Red-like operon (BAS) from Pseudomonas aeruginosa phage Ab31 and a Rac bacteriophage RecET-like operon (Rec-TEPsy) from P. syringae pv. syringae B728a and also contains exogenous elements, including the RecBCD inhibitor (Redγ or Pluγ) or single-stranded DNA-binding protein (SSB), that were added to enhance the PEHR recombineering efficiency. To solve the problem of false positives in Pseudomonas editing with the PEHR system, the processive enzyme Cas3 with a minimal Type I-C Cascade-based system was combined with PEHR. This protocol describes the utilization of a Pseudomonas-specific PEHR–Cas3 system that was designed to universally and proficiently modify the genomes of Pseudomonas species. The pipeline uses standardized cassettes combined with the concerted use of SacB counterselection and Cre site-specific recombinase for markerless or seamless genome modification, in association with vectors that possess the selectively replicating template R6K to minimize recombineering background. Compared with the traditional allelic exchange editing method, the PEHR–Cas3 system does not need to construct suicide plasmids carrying long homologous arms, thus simplifying the experimental procedure and shortening the traceless editing period. Compared with general editing systems based on phage recombinases, the PEHR–Cas3 system can effectively improve the screening efficiency of mutants using the cutting ability of Cas3 protein. The entire procedure requires ~12 days.

Key points

  • This protocol uses phage-encoded homologous recombination combined with Cascade–Cas3 for two- or three-step seamless genome modification in Pseudomonas, creating deletions, insertions or single-nucleotide substitutions. The authors also describe how to optimize the procedure for further Pseudomonas strains.

  • Compared with the traditional allelic exchange approach, the phage-encoded homologous recombination–Cas3 system provides a simpler and faster editing procedure, and the inclusion of Cas3 also improves recombineering accuracy.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Workflow for precision engineering the Pseudomonas genome with the PEHR–Cascade–Cas3 system (PEHR–Cas3).
Fig. 2: Seamless mutation using two-step recombination with the optimal recombination system.
Fig. 3: Seamless mutation using three-step modification with the optimal recombination system.
Fig. 4: Recombineering system optimization and genome editing in a new Pseudomonas strain.

Similar content being viewed by others

Data availability

Data shown in Figs. 2–4 as examples or anticipated results are available in the original papers7,28. Supplementary Figs. 1–5 are unpublished data, and the raw data behind the graphs are provided as Supplementary Data 1. Other supporting data are available upon reasonable request to the corresponding author.

References

  1. Palleroni, N. J. in Bergey’s Manual of Systematics of Archaea and Bacteria (eds. W. B. Whitman et al.) (2015).

  2. Hmelo, L. R. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat. Protoc. 10, 1820–1841 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Xin, X.-F., Kvitko, B. & He, S. Y. Pseudomonas syringae: what it takes to be a pathogen. Nat. Rev. Microbiol. 16, 316 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Choi, K. R., Cho, J. S., Cho, I. J., Park, D. & Lee, S. Y. Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida. Metab. Eng. 47, 463–474 (2018).

    CAS  PubMed  Google Scholar 

  5. Paulsen, I. T. et al. Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat. Biotechnol. 23, 873–878 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 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).

    CAS  PubMed  Google Scholar 

  7. Yin, J. et al. Single-stranded DNA-binding protein and exogenous RecBCD inhibitors enhance phage-derived homologous recombination in Pseudomonas. iScience 14, 1–14 (2019).

    PubMed  PubMed Central  Google Scholar 

  8. Yu, F. et al. Recombineering Pseudomonas protegens CHA0: an innovative approach that improves nitrogen fixation with impressive bactericidal potency. Microbiol. Res. 218, 58–65 (2019).

    CAS  PubMed  Google Scholar 

  9. Jing, X. et al. Engineering Pseudomonas protegens Pf-5 to improve its antifungal activity and nitrogen fixation. Microb. Biotechnol. 13, 118–133 (2020).

    CAS  PubMed  Google Scholar 

  10. Swingle, B., Bao, Z., Markel, E., Chambers, A. & Cartinhour, S. Recombineering using RecTE from Pseudomonas syringae. Appl. Environ. Microbiol. 76, 4960–4968 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Bao, Z., Cartinhour, S. & Swingle, B. Substrate and target sequence length influence RecTEPsy recombineering efficiency in Pseudomonas syringae. PLoS One 7, e50617 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Quenee, L., Lamotte, D. & Polack, B. Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in Pseudomonas aeruginosa. Biotechniques 38, 63–67 (2005).

    CAS  PubMed  Google Scholar 

  13. Gay, P., Le Coq, D., Steinmetz, M., Berkelman, T. & Kado, C. I. Positive selection procedure for entrapment of insertion sequence elements in Gram-negative bacteria. J. Bacteriol. 164, 918–921 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Li, R. et al. Development and application of an efficient recombineering system for Burkholderia glumae and Burkholderia plantarii. Microb. Biotechnol. 14, 1809–1826 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Aparicio, T., de Lorenzo, V. & Martinez-Garcia, E. CRISPR/Cas9-enhanced ssDNA recombineering for Pseudomonas putida. Microb. Biotechnol. 12, 1076–1089 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Czajka, J. J. et al. Tuning a high performing multiplexed-CRISPRi Pseudomonas putida strain to further enhance indigoidine production. Metab. Eng. Commun. 15, e00206 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Vareechon, C., Zmina, S. E., Karmakar, M., Pearlman, E. & Rietsch, A. Pseudomonas aeruginosa effector ExoS inhibits ROS production in human neutrophils. Cell Host Microbe 21, 611–618 (2017).

    Google Scholar 

  18. Rangel, S. M., Diaz, M. H., Knoten, C. A., Zhang, A. & Hauser, A. R. The role of ExoS in dissemination of Pseudomonas aeruginosa during pneumonia. PLoS Pathog. 11, 681 (2015).

    Google Scholar 

  19. Cimermancic, P. et al. Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters. Cell 158, 412–421 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Zheng, W. et al. Recombineering facilitates the discovery of natural product biosynthetic pathways in Pseudomonas parafulva. Biotechnol. J. 16, e2000575 (2021).

    PubMed  Google Scholar 

  21. Wang, X. et al. Improved dsDNA recombineering enables versatile multiplex genome engineering of kilobase-scale sequences in diverse bacteria. Nucleic Acids Res. 50, e15 (2022).

    CAS  PubMed  Google Scholar 

  22. Oliver, A., Mulet, X., Lopez-Causape, C. & Juan, C. The increasing threat of Pseudomonas aeruginosa high-risk clones. Drug Resist. Updat. 21–22, 41–59 (2015).

    PubMed  Google Scholar 

  23. Santajit, S. & Indrawattana, N. Mechanisms of antimicrobial resistance in ESKAPE pathogens. Biomed. Res. Int. 2016, 2475067 (2016).

    PubMed  PubMed Central  Google Scholar 

  24. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Hille, F. et al. The biology of CRISPR–Cas: backward and forward. Cell 172, 1239–1259 (2018).

    CAS  PubMed  Google Scholar 

  26. van Belkum, A. et al. Phylogenetic distribution of CRISPR–Cas systems in antibiotic-resistant Pseudomonas aeruginosa. mBio 6, e01796–01715 (2015).

    PubMed  PubMed Central  Google Scholar 

  27. Csorgo, B. et al. A compact Cascade–Cas3 system for targeted genome engineering. Nat. Methods 17, 1183–1190 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. W, Z. et al. Cascade–Cas3 facilitates high accuracy of genome engineering in Pseudomonas using phage-encoded homologous recombination. Eng. Microbiol. 2, 10046 (2022).

    Google Scholar 

  29. Zhang, Y., Buchholz, F., Muyrers, J. P. P. & Stewart, A. F. A new logic for DNA engineering using recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).

    CAS  PubMed  Google Scholar 

  30. Zhang, Y., Muyrers, J. P. P., Testa, G. & Stewart, A. F. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).

    CAS  PubMed  Google Scholar 

  31. Fu, J. et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat. Biotechnol. 30, 440–446 (2012).

    CAS  PubMed  Google Scholar 

  32. Yin, J. et al. A new recombineering system for Photorhabdus and Xenorhabdus. Nucleic Acids Res. 43, e36 (2015).

    PubMed  Google Scholar 

  33. Fels, U., Gevaert, K. & Van Damme, P. Bacterial genetic engineering by means of recombineering for reverse genetics. Front. Microbiol. 11, 548410 (2020).

    PubMed  PubMed Central  Google Scholar 

  34. Bunny, K., Liu, J. & Roth, J. Phenotypes of lexA Mutations in Salmonella enterica: evidence for a lethal lexa null phenotype due to the Fels-2 prophage. J. Bacteriol. 184, 6235–6249 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Beloin, C., Deighan, P., Doyle, M. & Dorman, C. J. Shigella flexneri 2a strain 2457T expresses three members of the H-NS-like protein family: characterization of the Sfh protein. Mol. Genet. Genom. 270, 66–77 (2003).

    CAS  Google Scholar 

  36. Derbise, A., Lesic, B., Dacheux, D., Ghigo, J. M. & Carniel, E. A rapid and simple method for inactivating chromosomal genes in Yersinia. FEMS Immunol. Med. Microbiol. 38, 113–116 (2003).

    CAS  PubMed  Google Scholar 

  37. Rossi, M.-S., Paquelin, A., Ghigo, J. M. & Wandersman, C. Haemophore-mediated signal transduction across the bacterial cell envelope in Serratia marcescens: the inducer and the transported substrate are different molecules. Mol. Microbiol. 48, 1467–1480 (2003).

    CAS  PubMed  Google Scholar 

  38. Hu, S. et al. Genome engineering of Agrobacterium tumefaciens using the lambda Red recombination system. Appl. Environ. Microbiol. 98, 2165–2172 (2014).

    CAS  Google Scholar 

  39. Egan, M., Ramirez, J., Xander, C., Upreti, C. & Bhatt, S. Lambda Red-mediated recombineering in the attaching and effacing pathogen Escherichia albertii. Biol. Proc. Online 18, 3 (2016).

    Google Scholar 

  40. Wannier, T. M. et al. Improved bacterial recombineering by parallelized protein discovery. Proc. Natl Acad. Sci. USA 117, 13689–13698 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Van Kessel, J. C. & Hatfull, G. F. Recombineering in Mycobacterium tuberculosis. Nat. Methods 4, 147–152 (2006).

    PubMed  Google Scholar 

  42. Pijkeren, J. P., Neoh, K. M., Sirias, D., Findley, A. S. & Britton, R. A. Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri. Bioengineered 3, 209–217 (2012).

    PubMed  PubMed Central  Google Scholar 

  43. Pijkeren, J. P. & Britton, R. A. High efficiency recombineering in lactic acid bacteria. Nucleic Acids Res. 40, e76 (2012).

    PubMed  PubMed Central  Google Scholar 

  44. Dong, H., Tao, W., Gong, F., Li, Y. & Zhang, Y. A functional recT gene for recombineering of Clostridium. J. Biotechnol. 173, 65–67 (2013).

    PubMed  Google Scholar 

  45. Sun, Z. et al. A high-efficiency recombineering system with PCR-based ssDNA in Bacillus subtilis mediated by the native phage recombinase GP35. Appl. Environ. Microbiol. 99, 5151–5162 (2015).

    CAS  Google Scholar 

  46. Yang, P., Wang, J. & Qi, Q. Prophage recombinases-mediated genome engineering in Lactobacillus plantarum. Microb. Cell Fact. 14, 154 (2015).

    PubMed  PubMed Central  Google Scholar 

  47. Bian, Z. et al. Development of a new recombineering system for Agrobacterium species. Appl. Environ. Microbiol. 88, e0249921 (2022).

    PubMed  Google Scholar 

  48. Cook, T. B. et al. Genetic tools for reliable gene expression and recombineering in Pseudomonas putida. J. Ind. Microbiol. Biotechnol. 45, 517–527 (2018).

    CAS  PubMed  Google Scholar 

  49. Chen, Z., Ling, W. & Shang, G. Recombineering and I-SceI-mediated Pseudomonas putida KT2440 scarless gene deletion. FEMS Microbiol. Lett. 363, fnw231 (2016).

    PubMed  Google Scholar 

  50. Luo, X. et al. Pseudomonas putida KT2440 markerless gene deletion using a combination of lambda Red recombineering and Cre/loxP site-specific recombination. FEMS Microbiol. Lett. 363, fnw014 (2016).

    PubMed  Google Scholar 

  51. Liang, R. & Liu, J. Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions. BMC Microbiol. 10, 209–209 (2010).

    PubMed  PubMed Central  Google Scholar 

  52. Aparicio, T., Jensen, S. I., Nielsen, A. T., de Lorenzo, V. & Martínez-García, 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–EM1319 (2016).

    CAS  PubMed  Google Scholar 

  53. Aparicio, T., de Lorenzo, V. & Martínez-García, E. CRISPR/Cas9-based counterselection boosts recombineering efficiency in Pseudomonas putida. Biotechnol. J. 13, 1700161 (2018).

    Google Scholar 

  54. Ricaurte, D. E. et al. A standardized workflow for surveying recombinases expands bacterial genome-editing capabilities. Microb. Biotechnol. 11, 176–188 (2018).

    CAS  PubMed  Google Scholar 

  55. Velazquez, E., Al-Ramahi, Y. & de Lorenzo, V. CRISPR/Cas9-enhanced targetron insertion for delivery of heterologous sequences into the genome of Gram-negative bacteria. Curr. Protoc. 2, e532 (2022).

    CAS  PubMed  Google Scholar 

  56. 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).

    CAS  PubMed  Google Scholar 

  57. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Yue, S. J. et al. Developing a CRISPR-assisted base-editing system for genome engineering of Pseudomonas chlororaphis. Microb. Biotechnol. 15, 2324–2336 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Volke, D. C., Friis, L., Wirth, N. T., Turlin, J. & Nikel, P. I. Synthetic control of plasmid replication enables target- and self-curing of vectors and expedites genome engineering of Pseudomonas putida. Metab. Eng. Commun. 10, e00126 (2020).

    PubMed  PubMed Central  Google Scholar 

  60. 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).

    CAS  PubMed  Google Scholar 

  61. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Qiu, D., Damron, F. H., Mima, T., Schweizer, H. P. & Yu, H. D. PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria. Appl. Environ. Microbiol. 74, 7422–7426 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Wang, F. et al. BrlR from Pseudomonas aeruginosa is a receptor for both cyclic di-GMP and pyocyanin. Nat. Commun. 9, 2563 (2018).

    PubMed  PubMed Central  Google Scholar 

  64. Jacobs, M. A. et al. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 100, 14339–14344 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Lee, S. A. et al. General and condition-specific essential functions of Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 112, 5189–5194 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240–2246 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Sharan, S. K., Thomason, L. C., Kuznetsov, S. G. & Court, D. L. Recombineering: a homologous recombination-based method of genetic engineering. Nat. Protoc. 4, 206–223 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Fu, J., Teucher, M., Anastassiadis, K., Skarnes, W. & Stewart, A.F. in Methods in Enzymology, 477. (eds. M. W. Paul & M. S. Philippe) 125–144 (Academic Press, 2010).

  69. Egan, S. M. & Schleif, R. F. DNA-dependent renaturation of an insoluble DNA binding protein. J. Mol. Biol. 243, 821–829 (1994).

    CAS  PubMed  Google Scholar 

  70. Egan, S. M. & Schleif, R. F. A regulatory cascade in the induction of rhaBAD. J. Mol. Biol. 234, 87–98 (1993).

    CAS  PubMed  Google Scholar 

  71. Filutowicz, M., McEachern, M. J. & Helinski, D. R. Positive and negative roles of an initiator protein at an origin of replication. Proc. Natl Acad. Sci. USA 83, 9645–9649 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Yang, S. H. et al. The SOSS1 single‐stranded DNA binding complex promotes DNA end resection in concert with Exo1. EMBO J. 32, 126–139 (2013).

    CAS  PubMed  Google Scholar 

  73. Pelicic, V., Reyrat, J. M. & Gicquel, B. Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J. Bacteriol. 178, 1197–1199 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Zhang, Z. & Lutz, B. Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein. Nucleic Acids Res. 30, e90–e90 (2002).

    PubMed  PubMed Central  Google Scholar 

  75. Mei, J., Benashski, S. & Firshein, W. Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli. J. Bacteriol. 177, 6766–6772 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Wang, H. L. et al. RecET direct cloning and Red alpha beta recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression. Nat. Protoc. 11, 1175–1190 (2016).

    CAS  PubMed  Google Scholar 

  77. Iwasaki, K. et al. Transformation of Pseudomonas putida by electroporation. Biosci. Biotechnol. Biochem. 58, 851–854 (1994).

    CAS  PubMed  Google Scholar 

  78. Wang, Q. et al. Quick and efficient method for genetic transformation of biopolymer-producing bacteria. J. Chem. Technol. Biot. 85, 775–778 (2010).

    CAS  Google Scholar 

  79. Kim, J. & Park, W. Oxidative stress response in Pseudomonas putida. Appl. Microbiol. Biotechnol. 98, 6933–6946 (2014).

    CAS  PubMed  Google Scholar 

  80. Sriwiriyarat, T., Jangkorn, S., Charoenpanich, J., Chinwetkitvanich, S. & Fongsatitkul, P. Occurrence of aerobic denitrifying bacteria in integrated fixed film activated sludge system. Chemosphere 285, 131504 (2021).

    CAS  PubMed  Google Scholar 

  81. Wang, H. et al. ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. Nucleic Acids Res. 46, e28 (2018).

    PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by grants from the National Key R&D Program of China (2019YFA0904000), the National Natural Science Foundation of China (31570094, 31670097 and 81502962); the 111 Project (B16030), the China Postdoctoral Science Foundation (2022M711925) to W.Z., the Shandong Provincial Natural Science Foundation of China (ZR2020MC015) to R.L. and (ZR2022QC107) to W.Z., the Guangdong Basic and Applied Basic Research Foundation (2022A1515110795) to W.Z., the Natural Science Foundation of Changsha (kq2208167) to J.Y., the Natural Science Foundation for Distinguished Young Scholars of Hunan Province (2023JJ10029) to J.Y. and the Taishan Scholar Program of Shandong Province to J.F.

Author information

Author notes

  1. These authors contributed equally: Wentao Zheng, Yandong Xia.

Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China

    Wentao Zheng, Xue Wang, Shiqing Gao, Vinothkannan Ravichandran, Chanjuan Jiang, Qiang Tu, Youming Zhang, Jun Fu & Ruijuan Li

  2. Hunan Provincial Key Laboratory of Animal Intestinal Function and Regulation, Hunan International Joint Laboratory of Animal Intestinal Ecology and Health, College of Life Sciences, Hunan Normal University, Changsha, China

    Yandong Xia, Diao Zhou, Yulong Yin & Jia Yin

  3. College of Life Science and Technology, Key Laboratory of National Forestry and Grassland Administration on Control of Artificial Forest Diseases and Pests in South China, Hunan Provincial Key Laboratory for Control of Forest Diseases and Pests, Key Laboratory for Non-wood Forest Cultivation and Conservation of Ministry of Education, Central South University of Forestry and Technology, Changsha, China

    Yandong Xia

  4. Chinese Academy of Sciences (CAS) Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Qiang Tu & Youming Zhang

Authors
  1. Wentao Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yandong Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xue Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shiqing Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Diao Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Vinothkannan Ravichandran
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chanjuan Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Qiang Tu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yulong Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Youming Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jun Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ruijuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Jia Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.Z., Y.X., Y.Y., Y.Z., J.F., R.L. and J.Y. designed the experiments. W.Z., Y.X., X.W., S.G., D.Z., C.C., V.R., C.J. and Q.T. performed the experiments. W.Z., Y.X., J.F., R.L. and J.Y. wrote the manuscript with help from all authors.

Corresponding authors

Correspondence to Jun Fu, Ruijuan Li or Jia Yin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks Thomas Eng, Kenan Murphy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Yin, J. et al. iScience 14, 1–14 (2019): https://doi.org/10.1016/j.isci.2019.03.007

Zheng, W. et al. Biotechnol. J. 16, e2000575 (2021): https://doi.org/10.1002/biot.202000575

Zheng, W. et al. Eng. Microbiol. 2, 10046 (2022): https://doi.org/10.1016/j.engmic.2022.100046

Supplementary information

Supplementary Information

Supplementary Tables 1–4, Figs. 1–5 and Notes 1–27.

Supplementary Data 1

Statistical source data for Supplementary Figs. 1, 2 and 5.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zheng, W., Xia, Y., Wang, X. et al. Precise genome engineering in Pseudomonas using phage-encoded homologous recombination and the Cascade–Cas3 system. Nat Protoc 18, 2642–2670 (2023). https://doi.org/10.1038/s41596-023-00856-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-023-00856-1

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research