λ Recombination and Recombineering

Abstract

The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics.

CRISPR /Cas9 recombineering‐mediated deep mutational scanning of essential genes in Escherichia coli

“…So, as the distance between the SPM and targeted mutation increases, the chances of decoupling of the target mutations and the SPM increase to introduce a PAM‐proximal bias (Fig ). According to this mechanism, the replication machinery uses the recombination donor as a template for replication due to which the mutations in the donor are not identified as mismatches (Murphy, ). This could explain why the mutation bias due to MMR is not observed.…”

Genetic Engineering by DNA Recombineering

“…In general, sequencing primers should be ß200 bp away from the homology arms so that any PCR products generated of the region will be at least 400 bp in length (Murphy, 2016;Thomason et al, 2014). PCR products shorter than 400 bp can be difficult to visualize on and purify from a standard agarose gel.…”

“…Because only about 50 base pairs (bp) of homology are required to facilitate recombination, the homology arms of a recombineering targeting cassette can be conveniently introduced as primer overhangs in a single PCR step (Murphy, 2016). The method utilizes the temporary expression of lambda phage genes (red αβγ ) that facilitate recombination between the target BAC or target genome and a DNA-targeting cassette that is introduced into the cell through electroporation.…”

“…The method utilizes the temporary expression of lambda phage genes (red αβγ ) that facilitate recombination between the target BAC or target genome and a DNA-targeting cassette that is introduced into the cell through electroporation. Because only about 50 base pairs (bp) of homology are required to facilitate recombination, the homology arms of a recombineering targeting cassette can be conveniently introduced as primer overhangs in a single PCR step (Murphy, 2016). Other homologous recombination-based methods generally require 500-to 1000-bp homology arms that need to be appended to a targeting cassette through time-consuming restriction cloning or difficult overlap-extension PCR (Hamilton, Aldea, Washburn, Babitzke, & Kushner, 1989;Kong, Yang, & Geller, 1999;Uil et al, 2011;Winans, Elledge, Krueger, & Walker, 1985).…”

“…The modified genomes can then produce genetically engineered virions in a process known as "viral rescue." Recombineering is also ideal for editing BACs with large fragments of human or animal genomes, such that these modified fragments can then be used as targeting cassettes to modify human or animal cells via non-recombineering homologous recombination (Murphy, 2016). Note that in human and animal cells, long homology arms are required, and thus targeting cassettes must necessarily be large (Baker et al, 2017;Hasty, Rivera-Pérez, & Bradley, 1991).…”