Orthogonal LoxPsym sites allow multiplexed site-specific recombination in prokaryotic and eukaryotic hosts

Charlotte Cautereels, Jolien Smets, Jonas De Saeger, Lloyd Cool, Yanmei Zhu, Anna Zimmermann, Jan Steensels, Anton Gorkovskiy, Thomas B. Jacobs and Kevin J. Verstrepen ()
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Charlotte Cautereels: VIB-KU Leuven Center for Microbiology
Jolien Smets: VIB-KU Leuven Center for Microbiology
Jonas De Saeger: Ghent University
Lloyd Cool: VIB-KU Leuven Center for Microbiology
Yanmei Zhu: VIB-KU Leuven Center for Microbiology
Anna Zimmermann: VIB-KU Leuven Center for Microbiology
Jan Steensels: VIB-KU Leuven Center for Microbiology
Anton Gorkovskiy: VIB-KU Leuven Center for Microbiology
Thomas B. Jacobs: Ghent University
Kevin J. Verstrepen: VIB-KU Leuven Center for Microbiology

Nature Communications, 2024, vol. 15, issue 1, 1-15

Abstract: Abstract Site-specific recombinases such as the Cre-LoxP system are routinely used for genome engineering in both prokaryotes and eukaryotes. Importantly, recombinases complement the CRISPR-Cas toolbox and provide the additional benefit of high-efficiency DNA editing without generating toxic DNA double-strand breaks, allowing multiple recombination events at the same time. However, only a handful of independent, orthogonal recombination systems are available, limiting their use in more complex applications that require multiple specific recombination events, such as metabolic engineering and genetic circuits. To address this shortcoming, we develop 63 symmetrical LoxP variants and test 1192 pairwise combinations to determine their cross-reactivity and specificity upon Cre activation. Ultimately, we establish a set of 16 orthogonal LoxPsym variants and demonstrate their use for multiplexed genome engineering in both prokaryotes (E. coli) and eukaryotes (S. cerevisiae and Z. mays). Together, this work yields a significant expansion of the Cre-LoxP toolbox for genome editing, metabolic engineering and other controlled recombination events, and provides insights into the Cre-LoxP recombination process.

Date: 2024
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DOI: 10.1038/s41467-024-44996-8

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