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Coupling ssDNA recombineering with CRISPR-Cas9 for Escherichia coli DnaG mutations

  • Jing Li
  • Jian Sun
  • Xinyue Gao
  • Zhixin Wu
  • Guangdong ShangEmail author
Methods and protocols

Abstract

Homologous recombination-based recombineering is a widely used DNA cloning and modification technique; recombineering efficiency improvement would be helpful for high-throughput DNA manipulation. Escherichia coli primase DnaG variants, such as DnaG Q576A and DnaG K580A, increase the recombineering efficiency via impairment of the interaction between primase and the replisome and boost the loading of more ssDNA on the replication fork. Bacterial adaptive immunity origin CRISPR-Cas9 is emerging as a powerful genome editing strategy. In this study, ssDNA recombineering and CRISPR-Cas9 were combined for the generation of DnaG variants. The tightly regulated Red operon expression cassette and tightly regulated Cas9 expression cassette were integrated into one chloroamphenicol resistance, p15A replicon-based vector. A self-curing, kanamycin resistance, p15A replicon-based plasmid was applied for the plasmid elimination after genome editing. The genome editing efficiency was as high as 100%. The recombineering efficiency of the strains harboring the DnaG variants was assayed via the kanamycin resistance gene repair as well as the chromosomal gene deletion experiments. The established genome editing strategy will expedite the DnaG structure and function relationship study as well as the metabolic engineering and synthetic biology applications.

Keywords

ssDNA recombineering CRISPR-Cas9 DnaG Plasmid curing 

Notes

Acknowledgements

We thank Dr. John Cronan, Dr. Thomas Kuhlman, and Dr. Barry Wanner for kindly providing the plasmids in this research. Funding was supported by the National Natural Science Foundation of China (NSFC 81273412).

Compliance with ethical standards

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2019_9744_MOESM1_ESM.pdf (782 kb)
ESM 1 (PDF 782 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life SciencesNanjing Normal UniversityNanjingChina

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