A Novel One-Step Method for Targeted Multiplication of DNA Fragments in Escherichia coli chromosome

E. coli is a well-characterized bacterium widely used as a workhorse in industrial biotechnology, synthetic biology, and metabolic engineering. Therefore, a wide variety of tools for E. coli chromosome editing was developed. However, the development of “easy-to-use” methods of insertion of the large chromosomal fragments into specific loci in multiple copies is still of current importance.

The CRISPR-Cas9 based methods are straightforward, efficient and reliable for making a single nucleotide mutations or deletions (Jiang, 2013). However, in case of targeted insertion of DNA fragments, CRISPR-Cas9-assisted tools require plasmid which will be a donor of genes for insertion (Jiang, 2015). The construction of a donor plasmid may be laborious and time-consuming task, especially in case of a long-length DNA fragment and/or a synthetic construct which expression as part of a plasmid is strongly undesirable.

We designed a novel technique for E. coli chromosome editing, which allows a targeted stable multiplication of a chosen long chromosome fragment in one-step experiment: the chromosome region of interest is in vivo subcloned via λRed-recombination into the short synthetic non-replicable DNA fragment containing the excisable marker and φ80-attP site. The resulting circle DNA molecule is immediately inserted into alternative chromosomal locus due to φ80-Integrase activity; the possibility for integration into several pre-existed φ80-attB sites simultaneously was demonstrated. In the proposed method, multiple ϕ80-attB recombination sites can be introduced into a recipient strain with CRISPR-Cas9 methodology.

Jiang, Wenyan, et al. "RNA-guided editing of bacterial genomes using CRISPR-Cas systems." Nature biotechnology 31.3 (2013): 233.

Jiang, Yu, et al. "Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system." Appl. Environ. Microbiol. 81.7 (2015): 2506-2514.