Reporting off-target effects of recombinant engineering using the pORTMAGE system

Brittany R Sanders; Sydney E Townsend; Maria L Ford; Joseph L Graves Jr; Misty D Thomas

doi:10.1016/j.mimet.2022.106627

Reporting off-target effects of recombinant engineering using the pORTMAGE system

J Microbiol Methods. 2023 Jan:204:106627. doi: 10.1016/j.mimet.2022.106627. Epub 2022 Nov 25.

Authors

Brittany R Sanders¹, Sydney E Townsend¹, Maria L Ford¹, Joseph L Graves Jr¹, Misty D Thomas²

Affiliations

¹ North Carolina Agricultural and Technical State University, USA.
² North Carolina Agricultural and Technical State University, USA. Electronic address: mthomas1@ncat.edu.

Abstract

pORTMAGE recombineering is a simple technique for incorporation of novel point mutations into bacterial genomes that eliminates off-target effects. Here we inserted point mutations into the cusS gene from Escherichia coli, then, using Illumina sequencing, report genetic variants in all mutant strains. Several off-site mutations were found at high frequency. Low frequency mutations also show high heterogeneity. This means that it is essential for studies to report all off-target effects and acknowledge the effect that this may have on resultant phenotypes.

Keywords: Off-target; Recombinant engineering; pORTMAGE.

Publication types

Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Escherichia coli* / genetics
Escherichia coli* / metabolism
Genetic Engineering* / methods
Genome, Bacterial
Mutation
Point Mutation

Grants and funding

T34 GM083980/GM/NIGMS NIH HHS/United States