CRMAGE: CRISPR Optimized MAGE Recombineering

TLDR

Genome engineering technologies are a bottleneck in metabolic engineering and systems biology. The study combines CRISPR/Cas9 with λ Red recombineering to develop a fast, highly efficient genome engineering method for E. coli. CRMAGE targets any genomic locus via degenerate codons, employs two USER‑cloned plasmids for rapid gRNA replacement, uses CRISPR/Cas9 to cure plasmids enabling multiple daily engineering rounds, and includes a web tool that predicts λ Red oligos and gRNAs.

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. 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.