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Targeted gene inactivation in Salmonella Typhi by CRISPR/Cas9-assisted homologous recombination

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Abstract

Background

Targeted gene inactivation (TGI) is a widely used technique for the study of genes’ functions. There are many different methods for TGI, however, most of them are so complicated and time-consuming. New promising genetic engineering tools are developing for this purpose. In the present study, for the first time we disrupted a virulence gene from Salmonella enterica serovar Typhi (S. Typhi), located in the bacterial chromosome using CRISPR/Cas9 system and homology directed repair (HDR).

Methods

For this aim, pCas9 plasmid containing Cas9 enzyme and required proteins for homology directed recombination was transferred to S. Typhi by electroporation. On the other hand, a specific guide RNA (gRNA) was designed using CRISPOR online tool. Synthetic gRNA was cloned into pTargetF plasmid. Also, a DNA fragment (HDR fragment) was designed to incorporate into the bacterial chromosome following the cleavage of the bacterial genome by Cas9 enzyme. pTargetF containing gRNA and HDR fragment were co-transferred to S. Typhi containing pcas9 plasmid. The transformed bacteria were screened for recombination using PCR, restriction digestion and sequencing.

Results

The results of PCR, restriction digestion and sequencing showed the successful recombination of S. Typhi, in which the gidA gene is disrupted.

Conclusion

In the present study we aimed to develop a rapid and robust method for targeted gene inactivation in a bacterial species, S. Typhi. This procedure can be exploited for disruption of other Salmonella as well as other bacteria’s genes.

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Acknowledgements

This study is a part of Y.T. PhD thesis. Authors thanks the staff of pathobiology Department of Shahid Bahonar University of Kerman and Biology Research Center of Imam Hossein University.

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Authors and Affiliations

  1. Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran

    Yousof Tarverdizadeh, Mohammad Khalili & Mehdi Golchin

  2. Department of Epidemiology and Biostatics, Research Centre for Emerging and Reemerging Infectious Diseases, Pasteur Institute of Iran, Tehran, Iran

    Saber Esmaeili

  3. Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

    Gholamreza Ahmadian

  4. Biology Research Center, Faculty of Basic Sciences, Imam Hossein University, Tehran, Iran

    Abbas Hajizade

Authors
  1. Yousof Tarverdizadeh
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  2. Mohammad Khalili
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  3. Saber Esmaeili
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  4. Gholamreza Ahmadian
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  5. Mehdi Golchin
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  6. Abbas Hajizade
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Correspondence to Abbas Hajizade.

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Tarverdizadeh, Y., Khalili, M., Esmaeili, S. et al. Targeted gene inactivation in Salmonella Typhi by CRISPR/Cas9-assisted homologous recombination. World J Microbiol Biotechnol 39, 58 (2023). https://doi.org/10.1007/s11274-022-03504-0

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  • DOI: https://doi.org/10.1007/s11274-022-03504-0

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