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.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970 Hørsholm, Denmark.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Ronda
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970 Hørsholm, Denmark.
authors.forename: Lasse Ebdrup
authors.initials: LE
authors.lastname: Pedersen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970 Hørsholm, Denmark.
authors.forename: Morten O A
authors.initials: MO
authors.lastname: Sommer
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970 Hørsholm, Denmark.
authors.forename: Alex Toftgaard
authors.initials: AT
authors.lastname: Nielsen
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D016923:Cell Death; D005818:Genetic Engineering / Q000379:methods*; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D059372:Nucleotide Motifs / Q000235:genetics; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D011938:Rec A Recombinases / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; D011938:Rec A Recombinases; D051719:MutS DNA Mismatch-Binding Protein