Abstract
Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system has been widely used in gene editing of various organisms. However, food-grade gene editing systems in lactic acid bacteria are still preliminary. Red/ET-dependent homologous recombination or CRISPR-based systems have been developed to gene editing in Lactococcus lactis, but these methods are overall inefficient. In the present study, a recombinant system based on CRISPR/Cas9 technology combined with Red/ET was developed using the plasmid pMG36e derived from Lactococcus lactis. Then, the developed recombinant system was applied to Lactococcus lactis. Knockout efficiency was significantly higher using the developed system (91%). In addition, this system showed the potential to be used as a high-throughput method for hierarchical screening. Finally, a gene-edited strain was obtained, and no antibiotics or exogenous genes were introduced using the developed gene editing system. Thus, the efficient system in lactic acid bacteria was constructed and optimized.
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Acknowledgements
This research was funded by Opening Project of Key Laboratory of Safety Assessment of Genetically Modified Organism (Food Safety) from Ministry of Agriculture and Rural Affairs of the People's Republic of China (SAGM-Y-2017006), the Key Research and Development Program of Hebei Province (Grant No. 19227134D), the Science and Technology Research Projects of Colleges and Universities in Hebei province (Grant No. ZD2021059), and the Food Processing Discipline Group of Hebei Agricultural University (Grant No. 2021-08).
Funding
Fnding was provided by Opening Project of Key Laboratory of Safety Assessment of Genetically Modified Organism (Food Safety) from Ministry of Agriculture and Rural Affairs of the People's Republic of China (Grant No. SAGM-Y-2017006), the Key Research and Development Program of Hebei Province (Grant No. 19227134D), the Science and Technology Research Projects of Colleges and Universities in Hebei province (Grant No. ZD2021059), and the Food Processing Discipline Group of Hebei Agricultural University (Grant No. 2021-08).
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Zhou, Y., Song, F., Yang, H. et al. Construction of a food-grade gene editing system based on CRISPR-Cas9 and its application in Lactococcus lactis NZ9000. Biotechnol Lett 45, 955–966 (2023). https://doi.org/10.1007/s10529-023-03398-4
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DOI: https://doi.org/10.1007/s10529-023-03398-4