CRMAGE: CRISPR Optimized MAGE Recombineering

Sci Rep. 2016 Jan 22:6:19452. doi: 10.1038/srep19452.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Cell Death
  • Genetic Engineering / methods*
  • MutS DNA Mismatch-Binding Protein / metabolism
  • Mutagenesis, Insertional
  • Mutation / genetics
  • Nucleotide Motifs / genetics
  • Oligonucleotides / metabolism
  • Plasmids / genetics
  • Protein Biosynthesis
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Rec A Recombinases / metabolism

Substances

  • Oligonucleotides
  • RNA, Guide, CRISPR-Cas Systems
  • Rec A Recombinases
  • MutS DNA Mismatch-Binding Protein