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.

A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome

“…This resulted in a labor-intensive and time-consuming procedure, especially for large-scale multiplex or iterative whole-genome engineering9 (Table 2). The use of short single-stranded DNA (ssDNA) as templates for recombineering that is independent of the selective marker greatly facilitated whole-genome engineering32, as exemplified by multiplex automated genome engineering (MAGE)3334. However, the genetic modification range of ssDNA recombineering is constrained by the length of the ssDNA, and large-scale chromosomal DNA fragment deletion is therefore not possible using that method.…”

“…The CRISPR-Ca9 system has been broadly adopted in markerless genome engineering, and its utility and flexibility in genetic manipulation of the bacterial genome have improved dramatically34373839404142. Recently, Beier et al .…”

Increased production of L-serine in Escherichia coli through Adaptive Laboratory Evolution

“…Two key mutations lrp D143G and rho R87L were introduced in Q1(DE3) thrA S357R strain separately using the CRMAGE system (Ronda et al, 2016). The pMAZSK plasmids containing gRNA targeting wild type lrp and rho was constructed using primers shown in Table S5.…”

“…The PCR product was subsequently phosphorylated with polynucleotide kinase, ligated with T4 DNA ligase and transformed. The isolated plasmids were used for the CRMAGE protocol (Ronda et al, 2016), and the resulting mutants were sequenced for targeted loci (by gF and gR primers listed in Table S5) and subjected for growth profiling studies.…”

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli

“…Choi et al, T 2016;S. Choi et al, 2016;Park et al, 2011Park et al, , 2007Yim et al, 2011) owing to the rich toolset available for gene and expression modification (Datsenko and Wanner, 2000;Jiang et al, 2015;Ronda et al, 2016;Wang et al, 2009) as well as accumulated historical knowledge base of biochemical understanding (Guo et al, 2013;Kanehisa et al, 2017;Keseler et al, 2013;Monk et al, 2017). Seven commonly used strains in biotechnology applications include K-12 MG1655, K-12 W3110, K-12 DH5a, BL21, C, Crooks, and W. It has been shown that particular strains are better suited for different applications (Monk et al, 2016).…”