title: A Tool for Multiple Targeted Genome Deletions that Is Precise, Scar-Free, and Suitable for Automation.
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.
authors.affiliation: Department of Computer Science, Aberystwyth University, Aberystwyth, SY23 3DB, United Kingdom.
authors.forename: Wayne
authors.initials: W
authors.lastname: Aubrey
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, SY23 3DD, United Kingdom.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Riley
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, SY23 3DD, United Kingdom.
authors.forename: Michael
authors.initials: M
authors.lastname: Young
authors.affiliation: Manchester Institute of Biotechnology and School of Computer Science, University of Manchester, Manchester, M1 7DN, United Kingdom.
authors.forename: Ross D
authors.initials: RD
authors.lastname: King
authors.affiliation: Cambridge Systems Biology Centre and Department of Biochemistry, University of Cambridge, Sanger Building, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Oliver
authors.affiliation: Department of Computer Science, Aberystwyth University, Aberystwyth, SY23 3DB, United Kingdom.
authors.forename: Amanda
authors.initials: A
authors.lastname: Clare
mesh_terms: D001331:Automation; D002921:Cicatrix / Q000235:genetics*; D019295:Computational Biology / Q000379:methods*; D016681:Genome, Fungal; D023281:Genomics / Q000379:methods; D016366:Open Reading Frames / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D017384:Sequence Deletion; D012984:Software