Maxence Lejars: EASY-CRISPR: a toolbox for high-throughput single-step custom genetic editing in E. coli. Séminaire Externe MAP - invité par l'Equipe CRP

Maxence LEJARS, post-doctorant à l'IBCP, équipe RNA control of gene expression dirigée par Maude Guillier du laboratoire Microbial Gene Expression (EGM)- UMR8261 CNRS, Université Paris Cité.

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.

Maxence Lejars1, Tomoya Maeda2 and Maude Guillier1
1Microbial Gene Expression department, UMR8261 CNRS, Université Paris Cité, Institut de Biologie Physico-Chimique, Paris 75005, France
2Laboratory of Microbial Physiology, Research Faculty of Agriculture, Hokkaido University, Sapporo, Hokkaido 060-8589, Japan