title: Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli.
abstract: To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and "scar"-less.
authors.affiliation: Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Pyne
authors.affiliation: Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Murray
authors.initials: M
authors.lastname: Moo-Young
authors.affiliation: Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada Neemo Inc., Hamilton, Ontario, Canada duane.chung@uwaterloo.ca cpchou@uwaterloo.ca.
authors.forename: Duane A
authors.initials: DA
authors.lastname: Chung
authors.affiliation: Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada duane.chung@uwaterloo.ca cpchou@uwaterloo.ca.
authors.forename: C Perry
authors.initials: CP
authors.lastname: Chou
mesh_terms: D010582:Bacteriophage lambda / Q000201:enzymology; D064113:CRISPR-Cas Systems; D004926:Escherichia coli / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D016254:Mutagenesis, Insertional; D010957:Plasmids; D045522:Recombinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D017384:Sequence Deletion; D013297:Streptococcus pyogenes / Q000201:enzymology
chemical_list: D045522:Recombinases