Homologous recombination can be used to replace or knockout genes in bacteria, through lambda red and CRISPR-Cas recombineering systems.
In order to replace or knockout a gene using lambda red, the pKD46 plasmid is introduced into the target bacteria by transformation. This is a temperature-sensitive plasmid containing gam, beta, and exo (the three genes of the lambda red system), under an arabinose-inducible promoter (pBAD). This allows the expression of the lambda red system to be controlled to growth media containing arabinose. Exo is an exonuclease, degrading the dsDNA to ssDNA, with beta binding to the ssDNA to promote annealing with complementary DNA. Gam inhibits DNA repair enzymes, allowing homologous recombination to occur. If lambda red is being used to replace a gene, the gene will be introduced as a section of ssDNA. This will have a low transformation efficiency, as ssDNA is easily degraded by the bacterial cell (as part of a defence mechanism), and so electroporation will likely be used to give improved transformation efficiency. Regions of homology must be present either side of the gene, to allow homologous recombination to insert the gene (these can be added by PCR, using primers that contain an overhang with the homologous region in). This is due to the DNA having to anneal to the ssDNA formed by exo (and therefore must be complementary). Where a gene knockout is desired, no ssDNA will be introduced to the cell. Instead, the gene being knocked out will be removed and homologous recombination mechanisms used to re-join the ends together (by non-homologous end joining).
CRISPR-Cas is a highly specific gene editing tool used to modify genomes in vivo. Where a gene of interest is to be inserted into the host chromosome, regions of homology must be present either side. This allows site-specific recombination to be employed, where shorter homologous sections are needed (~40bp). Using a guide RNA (gRNA), the insertion of the gene of interest can be highly specific, allowing easy targeting when the genome of the organism is known (or the section of interest). To remove a gene with CRISPR-Cas, no gene of interest is provided. Instead, non-homologous end joining is used. Cas9 is targeted to the relevant sites to cleave the DNA, allowing the specific and selective modification of chromosomal DNA to knockout genes of interest.