Journal article
CRMAGE: CRISPR Optimized MAGE Recombineering
Bacterial Cell Factories, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
iLoop, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
Bacterial Synthetic Biology, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark5
Bacterial Cell Factory Optimization, Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark6
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.
Language: | English |
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Publisher: | Nature Publishing Group |
Year: | 2016 |
Pages: | 19452 |
ISSN: | 20452322 |
Types: | Journal article |
DOI: | 10.1038/srep19452 |
ORCIDs: | Pedersen, Lasse Ebdrup , Sommer, Morten Otto Alexander and Nielsen, Alex Toftgaard |