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

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 marke...

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Bibliographic Details
Published in:PloS one 2015-12, Vol.10 (12), p.e0142494
Main Authors: Aubrey, Wayne, Riley, Michael C, Young, Michael, King, Ross D, Oliver, Stephen G, Clare, Amanda
Format: Article
Language:English
Subjects:
Analysis
Automation
Binding sites
Cicatrix - genetics
Computational Biology - methods
Design for recycling
Gene sequencing
Genetic engineering
Genome, Fungal
Genomes
Genomic instability
Genomics
Genomics - methods
Markers
Mycoplasma genitalium
Oligonucleotides
Open reading frames
Open Reading Frames - genetics
Polymerase Chain Reaction
Primers
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Schizosaccharomyces - genetics
Sequence Deletion
Software
Stability
Synthetic biology
Yeast
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0142494