title: Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing.
abstract: The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair. However, E. coli lacks robust systems for double-strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9-mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ-Red system for recombination in E. coli, we created a highly efficient system for marker-free and scarless genome editing. © 2017 by John Wiley & Sons, Inc.
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.|Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Reisch
authors.affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Kristala L J
authors.initials: KLJ
authors.lastname: Prather
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics; D004720:Endonucleases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D011995:Recombination, Genetic; D014170:Transformation, Genetic
chemical_list: D001426:Bacterial Proteins; D004247:DNA; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR/Cas9; Escherichia coli; gene deletions; genome editing; metabolic engineering; λ-Red