A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

Ákos Nyerges, Bálint Csörgo, István Nagy, Balázs Bálint, Péter Bihari, Viktória Lázár, Gábor Apjok, Kinga Umenhoffer, Balázs Bogos, G. Pósfai, C. Pál

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.

Original languageEnglish
Pages (from-to)2502-2507
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume113
Issue number9
DOIs
Publication statusPublished - Mar 1 2016

Fingerprint

DNA Mismatch Repair
Genome
Escherichia coli
Bacterial Genomes
Salmonella enterica
Workflow
Mutant Proteins
Microbial Drug Resistance
Oligonucleotides
Mutagenesis
DNA Repair
Plasmids
Mutation
Therapeutics
Gene Editing

Keywords

  • Genome engineering
  • Methyl-directed mismatch repair
  • Off-target effects
  • Recombineering
  • Synthetic biology

ASJC Scopus subject areas

  • General

Cite this

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. / Nyerges, Ákos; Csörgo, Bálint; Nagy, István; Bálint, Balázs; Bihari, Péter; Lázár, Viktória; Apjok, Gábor; Umenhoffer, Kinga; Bogos, Balázs; Pósfai, G.; Pál, C.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 113, No. 9, 01.03.2016, p. 2502-2507.

Research output: Contribution to journalArticle

Nyerges, Ákos ; Csörgo, Bálint ; Nagy, István ; Bálint, Balázs ; Bihari, Péter ; Lázár, Viktória ; Apjok, Gábor ; Umenhoffer, Kinga ; Bogos, Balázs ; Pósfai, G. ; Pál, C. / A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species. In: Proceedings of the National Academy of Sciences of the United States of America. 2016 ; Vol. 113, No. 9. pp. 2502-2507.
@article{7f8c340dd4e6412f94571dd3cb378e11,
title = "A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species",
abstract = "Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.",
keywords = "Genome engineering, Methyl-directed mismatch repair, Off-target effects, Recombineering, Synthetic biology",
author = "{\'A}kos Nyerges and B{\'a}lint Cs{\"o}rgo and Istv{\'a}n Nagy and Bal{\'a}zs B{\'a}lint and P{\'e}ter Bihari and Vikt{\'o}ria L{\'a}z{\'a}r and G{\'a}bor Apjok and Kinga Umenhoffer and Bal{\'a}zs Bogos and G. P{\'o}sfai and C. P{\'a}l",
year = "2016",
month = "3",
day = "1",
doi = "10.1073/pnas.1520040113",
language = "English",
volume = "113",
pages = "2502--2507",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "9",

}

TY - JOUR

T1 - A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

AU - Nyerges, Ákos

AU - Csörgo, Bálint

AU - Nagy, István

AU - Bálint, Balázs

AU - Bihari, Péter

AU - Lázár, Viktória

AU - Apjok, Gábor

AU - Umenhoffer, Kinga

AU - Bogos, Balázs

AU - Pósfai, G.

AU - Pál, C.

PY - 2016/3/1

Y1 - 2016/3/1

N2 - Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.

AB - Currently available tools for multiplex bacterial genome engineering are optimized for a few laboratory model strains, demand extensive prior modification of the host strain, and lead to the accumulation of numerous off-target modifications. Building on prior development of multiplex automated genome engineering (MAGE), our work addresses these problems in a single framework. Using a dominant-negative mutant protein of the methyl-directed mismatch repair (MMR) system, we achieved a transient suppression of DNA repair in Escherichia coli, which is necessary for efficient oligonucleotide integration. By integrating all necessary components into a broad-host vector, we developed a new workflow we term pORTMAGE. It allows efficient modification of multiple loci, without any observable off-target mutagenesis and prior modification of the host genome. Because of the conserved nature of the bacterial MMR system, pORTMAGE simultaneously allows genome editing and mutant library generation in other biotechnologically and clinically relevant bacterial species. Finally, we applied pORTMAGE to study a set of antibiotic resistance-conferring mutations in Salmonella enterica and E. coli. Despite over 100 million y of divergence between the two species, mutational effects remained generally conserved. In sum, a single transformation of a pORTMAGE plasmid allows bacterial species of interest to become an efficient host for genome engineering. These advances pave the way toward biotechnological and therapeutic applications. Finally, pORTMAGE allows systematic comparison of mutational effects and epistasis across a wide range of bacterial species.

KW - Genome engineering

KW - Methyl-directed mismatch repair

KW - Off-target effects

KW - Recombineering

KW - Synthetic biology

UR - http://www.scopus.com/inward/record.url?scp=84959418965&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84959418965&partnerID=8YFLogxK

U2 - 10.1073/pnas.1520040113

DO - 10.1073/pnas.1520040113

M3 - Article

VL - 113

SP - 2502

EP - 2507

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 9

ER -