Lejars Maxence, Maeda Tomoya, Guillier Maude
Expression génétique microbienne, UMR8261 CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris 75005, France.
Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Hokkaido, Japan.
Nucleic Acids Res. 2025 Sep 5;53(17). doi: 10.1093/nar/gkaf883.
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: Engineered Assembly of SYnthetic operons for targeted editing (EASY-edit) 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-edit 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-edit 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.
使用CRISPR-Cas9辅助重组工程可以实现靶向基因编辑。然而，高效编辑需要针对每个要修饰的位点进行仔细优化，这可能既繁琐又耗时。在这项工作中，我们开发了一种简单、快速且廉价的方法：在大肠杆菌中用于靶向编辑的合成操纵子工程组装（EASY-edit）。通过使用一组优化的引导RNA和携带相对短同源臂的单链或双链DNA修复模板，可以实现对操纵子不同组成元件的高效编辑。这有助于构建多种遗传工具，包括突变体文库或报告基因。EASY-edit也是高度模块化的，因为我们提供了插入三个位点的操纵子的替代和互补版本，这些版本可以迭代编辑并轻松组合。作为概念验证，我们报告了与报告基因的几种融合构建，证实了已知的转录后调控机制，以及饱和且无偏倚的突变体文库的构建。总之，EASY-edit系统提供了一个灵活的基因组表达平台，可用于理解生物学过程以及作为生物工程应用的工具。
