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bioRxiv · 10.1101/2025.03.20.644322

EASY-CRISPR: a toolbox for high-throughput single-step custom genetic editing in bacteria

Lejars, M.·Maeda, T.·Guillier, M.
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Abstract

Targeted gene editing can be achieved using CRISPR/Cas9-assisted recombineering. However, high-efficiency editing requires careful optimization for each locus to be modified which can be tedious and time-consuming. In this work, we developed a simple, fast and cheap method for the Editing and Assembly of SYnthetic operons using CRISPR/Cas9-assisted recombineering (EASY-CRISPR) in Escherichia coli. Highly efficient editing of the different constitutive elements of the operons can be achieved by using a set of optimized guide RNAs and single- or double-stranded DNA repair templates carrying relatively short homology arms. This facilitates the construction of multiple genetic tools, including mutant libraries or reporter genes. EASY-CRISPR is also highly modular, as we provide alternative and complementary versions of the operon inserted in three loci which can be edited iteratively and easily combined. As a proof of concept, we report the construction of several fusions with reporter genes confirming known post-transcriptional regulation mechanisms and the construction of saturated and unbiased mutant libraries. In summary, the EASY-CRISPR system provides a flexible genomic expression platform that can be used both for the understanding of biological processes and as a tool for bioengineering applications.

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Lejars, M., Maeda, T., Guillier, M.. 2025-03-20. EASY-CRISPR: a toolbox for high-throughput single-step custom genetic editing in bacteria. https://doi.org/10.1101/2025.03.20.644322

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