1887

Abstract

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 -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.

Funding
This study was supported by the:
  • Deutsche Forschungsgemeinschaft (Award SCHN 371 / 11-1)
    • Principle Award Recipient: KarinSchnetz
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/content/journal/micro/10.1099/mic.0.001173
2022-04-12
2022-05-18
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