Deletion of FRT-sites by no-SCAR recombineering in Escherichia coli

Abstract

Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in Escherichia coli and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNAFRT, and locus-specific ssDNA oligonucleotides. We applied this method to construct a scarless E. coli strain suitable for gradual induction by l-arabinose. Genome sequencing of the resulting strain and its parent strains demonstrated that no additional mutations were introduced along with the simultaneous deletion of two FRT scar-sites. The FRT-specific no-SCAR selection by sgRNAFRT/Cas9 may be generally applicable to cure FRT scar-sites of E. coli strains constructed by classical λ-Red recombineering.

Mobile Translation Systems Generate Genomically Engineered Escherichia coli Cells with Improved Growth Phenotypes

“…Additionally, the Cre/LoxP recombination system could offer an alternative site-specific recombination system to FLP/FRT recombination. Furthermore, multiplexed automated genome engineering or no-scar recombineering might be useful for inserting or deactivating/deleting targeted FRT sites over the course of a mobilization experiment or after desired genome engineering is complete in order to produce an FRT-less strain. , Finally, high-throughput automation and selection could expand the power of mobilization to generate effective functional phenotypes.…”

Microbial musings – Summer 2022

“…The first E. coli paper to highlight is an enhancement of the standard Datsenko and Wanner method [20], which addresses the issue that when insertions are converted to unmarked deletions a small ‘scar’ sequence is left on the genome. When multiple deletions are made in the same strain the presence of these identical sequences can lead to large genomics rearrangements by homologous recombination, a problem we have seen in my lab in York.…”

“…When multiple deletions are made in the same strain the presence of these identical sequences can lead to large genomics rearrangements by homologous recombination, a problem we have seen in my lab in York. The development of orthogonal Flp recognition target (FRT) sites, which leave non-identical scar sites, partly solves this problem but requires more design considerations, so the authors of this paper, led by Aathmaja Anandhi Rangarajan (@Aathmaja) from the group of Karin Schnetz at the University of Cologne, Germany, have developed the 'no-SCAR' Cas9-assisted recombineering method for the deletion of the scar sites from the chromosome [20]. The method requires the use of a plasmid they developed, pKDsgRNA-FRT and a single-stranded oligonucleotide specific for the template, for the repair of the particular scar being removed.…”