CRMAGE: CRISPR Optimized MAGE Recombineering View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2016-05

AUTHORS

Carlotta Ronda, Lasse Ebdrup Pedersen, Morten O A Sommer, Alex Toftgaard Nielsen

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. More... »

PAGES

19452

References to SciGraph publications

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/srep19452

    DOI

    http://dx.doi.org/10.1038/srep19452

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1034208257

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/26797514


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