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.
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.
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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.
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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.
The authors declare no competing interests.
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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
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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