A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation.

Wayne Aubrey, Michael Riley, Michael Young, Ross Donald King, Stephen G. Oliver, Amanda Clare

Research output: Contribution to journalArticlepeer-review

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Abstract

Many advances in synthetic biology require the removal of a large number of genomic elements from a genome. Most existing deletion methods leave behind markers, and as there are a limited number of markers, such methods can only be applied a fixed number of times. Deletion methods that recycle markers generally are either imprecise (remove untargeted sequences), or leave scar sequences which can cause genome instability and rearrangements. No existing marker recycling method is automation-friendly. We have developed a novel openly available deletion tool that consists of: 1) a method for deleting genomic elements that can be repeatedly used without limit, is precise, scar-free, and suitable for automation; and 2) software to design the method’s primers. Our tool is sequence agnostic and could be used to delete large numbers of coding sequences, promoter regions, transcription factor binding sites, terminators, etc in a single genome. We have validated our tool on the deletion of non-essential open reading frames (ORFs) from S. cerevisiae. The tool is applicable to arbitrary genomes, and we provide primer sequences for the deletion of: 90% of the ORFs from the S. cerevisiae genome, 88% of the ORFs from S. pombe genome, and 85% of the ORFs from the L. lactis genome.
Original languageEnglish
Article numbere0142494
JournalPLoS One
Volume10
Issue number12
DOIs
Publication statusPublished - 02 Dec 2015

Keywords

  • PCR
  • synthetic biology
  • GENE DELETION
  • SACCHAROMYCES CEREVISIAE
  • automation
  • Sequence Deletion
  • Automation
  • Cicatrix/genetics
  • Open Reading Frames/genetics
  • Computational Biology/methods
  • Genomics/methods
  • Saccharomyces cerevisiae/genetics
  • Genome, Fungal
  • Polymerase Chain Reaction
  • Software
  • Schizosaccharomyces/genetics

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