4-Way Recombineering (4WR)

A New Application for E. coli GB05-dir

4-Way Recombineering (4WR) offers a new fast and streamlined way to assemble plasmids in a single in vivo recombination step from up to four DNA fragments, e.g. PCR products. This is facilitated by co-electroporation of all fragments to Gene Bridges’ popular E. coli stain GB05-dir (Cat. No. K008).

The 4WR system greatly complements existing technologies such as CRISPR/Cas9. In recent years CRISPR/Cas9 has become the system of choice for the editing of eukaryotic chromosomes. There are many elaborated strategies to address a genomic site by the guiding RNA and mutant Cas9 nucleases in a strikingly precise manner. But when it comes to allelic exchange, gene fusion, or protein tagging operations methods to construct corresponding donor vectors are still traditional, thus, time consuming and tricky. The power of 4-Way Recombineering was impressively demonstrated by Baker and coworkers (2016, Nature Scientific Reports 6, 25529), who used 4WR for the very rapid construction of donor vectors with short Homology Regions (~1 kb). Such constructs work remarkably efficient for mono-allelic applications, such as fluorescent protein tagging, targeting the male Y-chromosome, or heterozygous targeting. This combination of straightforward technologies was termed Recombineering And Cas9-assisted tagging (RAC-tagging).

The applications of 4WR also extends to laboratory standard operations like the cloning of whole plasmid series differing in a specific component, e.g. promoter libraries driving an invariant reporter gene, or exchanging of antibiotic resistance markers or protein tags. Because the 4WR is modular by design, implementation of such tasks is quite easy.

Together with the earlier developed Direct Cloning (DC) technique, the 4WR further allows for handling of large portions of DNA, e.g. whole gene clusters. Operons which were sub-cloned from genomic DNA in an initial DC step, can easily combined with non-native promoters or protein tags in subsequent 4WR operations.

You can find detailed order information on Gene Bridges' E. coli strain GB05-dir here.

Customers who already purchased the GB05-dir strain can find the new 4WR Application Note here.

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