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Microbes and microbial‐derived systems have been extensively used for the development of novel DNA engineering tools and for the application of these tools to DNA cloning. Restriction enzymes, recombinase systems such as CRE/Lox, integrases such as ΦC31‐Int, and the Cas9‐CRISPR system have all microbial origin. Recombinases and integrases‐based systems have been extensively used to engineer the mammalian genomes but we will not discuss them in this book that is focusing on scarless genome engineering systems. This chapter will focus on the development of Recombineering for bacterial engineering and its use in genome engineering with particular focus on applications in drug discovery.

The inspiration for the Recombineering (recombination‐mediated DNA engineering) method came from studies in yeast where the possibility to introduce an exogenous DNA cassette in yeast genome was demonstrated in the years 1978–1979. Yeasts DNA recombination methods used homology arms to target a gene without the need of double‐strand breaks (DSBs) generation or the expression of any exogenous protein (Hinnen et al. 1978; Scherer and Davis 1979). It was also demonstrated in yeasts that single‐stranded oligonucleotides with short homology arms to a target sequence are able to promote genomic insertions/deletions/modifications (Bhargava et al. 1999) Unfortunately, this approach does not work efficiently in wild‐type bacteria or in higher eukaryotes. This has limited the utility of single‐strand oligonucleotides‐mediated DNA editing of bacterial and mammalian genomes before the advent of recombineering. Murphy´s and Stewart´s groups showed for the first time that a portable recombination system can be introduced in Escherichia coli to induce recombination in bacteria in a similar fashion to the yeast recombination system but with much higher efficiency (Murphy 1998; Zhang et al. 1998). The portable cassette encodes for an exonuclease (i.e. Redα for the lambda Red system), a DNA annealing protein (i.e. Redβ), and the RecBCD inhibitor (i.e. Redγ). In particular, Stewart´s group showed that this system works with very short homology arms (as short as 30nt) via a peculiar mechanism of single‐strand heteroduplex intermediates at the replication fork (Maresca et al. 2010). This observation paved the way to the use of Recombineering for Precise Genome Editing of Bacterial Genome and for molecular cloning strategies. Recombineering overcomes the limitation of classical restriction/ligation‐based cloning because it does not require the availability of unique restriction sites in the target plasmid and it is specific enough to target the bacterial genome. Therefore, Recombineering has been extensively used for the seamless engineering of large constructs such as bacterial artificial chromosomes (BACs) and for the engineering of bacterial genome.

Figure 2.1 Graphical overview of genome engineering technologies (upper panel) and methods (lower panel) developed during the latest 27 years. A schematic representation of the different technologies or methods is presented, respectively, above or under the timeline arrow.