Deletion of FRT-sites by no-SCAR recombineering in .

Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in 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, sgRNA, and locus-specific ssDNA oligonucleotides. We applied this method to construct a scarless 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 sgRNA/Cas9 may be generally applicable to cure FRT scar-sites of strains constructed by classical λ-Red recombineering.