CRMAGE: CRISPR Optimized MAGE Recombineering

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Abstract

A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering.
Original languageEnglish
Article number19452
JournalScientific Reports
Volume6
Number of pages11
ISSN2045-2322
DOIs
Publication statusPublished - 2016

Cite this

@article{83974b0ef4224a168f44ceeafd6fcccf,
title = "CRMAGE: CRISPR Optimized MAGE Recombineering",
abstract = "A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5{\%} and 99.7{\%} for gene recoding of three genomic targets, compared to between 0.68{\%} and 5.4{\%} using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6{\%} to 70{\%}. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering.",
author = "Carlotta Ronda and Pedersen, {Lasse Ebdrup} and Sommer, {Morten Otto Alexander} and Nielsen, {Alex Toftgaard}",
year = "2016",
doi = "10.1038/srep19452",
language = "English",
volume = "6",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",

}

CRMAGE: CRISPR Optimized MAGE Recombineering. / Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten Otto Alexander; Nielsen, Alex Toftgaard.

In: Scientific Reports, Vol. 6, 19452, 2016.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - CRMAGE: CRISPR Optimized MAGE Recombineering

AU - Ronda, Carlotta

AU - Pedersen, Lasse Ebdrup

AU - Sommer, Morten Otto Alexander

AU - Nielsen, Alex Toftgaard

PY - 2016

Y1 - 2016

N2 - A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering.

AB - A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering.

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M3 - Journal article

VL - 6

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

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