Introduction:

Systematic reengineering of bacterial genomes is still in its infancy. Advances in genome engineering may enable novel applications in green biotechnology while simultaneously providing fundamental insights into the interplay between biological function and genome architecture.

Goals:

Here, we present a scalable in vivo method to iteratively relocate ("cut"n"paste") functional genomic loci onto artificial chromosomes in bacteria. Our method aims to facilitate the creation of auxiliary functional modules, such as the recently described iModulons¹, that can be transferred and used in a plug-and-play fashion to add specific biological functionality to a minimal cell chassis when desired.

Methods:

We combine CRISPR-Cas9 counter-selection and lambda red recombineering with standardised components and an antibiotic marker recycling scheme to drastically reduce the need for in vitro manipulations and locus-specific workflow optimization.

Results:

We successfully relocate multiple operons onto an artificial chromosome and assay their performance in the novel genomic context.

Summary:

In vivo "cut"n"paste" promises to overcome the stark limitations imposed by in vitro techniques such as PCR or restriction enzyme cloning by letting CRISPR-Cas9, homologous recombineering, and the native bacterial DNA polymerase do the heavy lifting inside the cell.

References:

Sastry, A.V., Gao, Y., Szubin, R. et al. The Escherichia coli transcriptome mostly consists of independently regulated modules. Nat Commun 10, 5536 (2019). https://doi.org/10.1038/s41467-019-13483-w