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

Wayne Aubrey,Amanda Clare,Ross D King,Michael C Riley,Michael Young,Stephen G Oliver

doi:10.1371/journal.pone.0142494

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.

Highlights

Techniques for the deletion of multiple genes and other genomic regions are a standard part of the modern biologist’s toolbox [1,2,3,4,5,6]
We have developed a deletion tool that consists of: 1) a method for deleting genomic elements that can be repeatedly used without limit and is precise, scar-free, and suitable for automation; and 2) software to design the primers necessary for the method
From the 96 test ORFs deleted, in a number of cases there was an unexpected lack of visible product

Summary

Introduction

Techniques for the deletion of multiple genes and other genomic regions are a standard part of the modern biologist’s toolbox [1,2,3,4,5,6]. There are many genome-wide mutagenesis methods which alter genomes by integrating transposable elements or synthetic oligonucleotides into cells [12,13,14]. The alterations they make are irrational and these. PLOS ONE | DOI:10.1371/journal.pone.0142494 December 2, 2015

Methods

Results

Conclusion