Recombineering: A Modern Approach to Genetic Engineering

Recombineering is a highly efficient and precise method for genetically engineering DNA in vivo. With recombineering one can make gene replacements, deletions, insertions, inversions, and single and multiple, point mutations. Gene cloning, in vivo plasmid construction, and gene/protein tagging are also possible. Recombineering is catalyzed by the bacteriophage λ Red, RecET, or similar phage-encoded recombination systems and can utilize both double- and single-stranded DNAs as substrates. All genetic alterations are precise to the base pair as designed by the user and are mediated efficiently by DNA containing target homologies of 30–50 bases. These homologies are short enough to be incorporated into commercially available oligonucleotides. Since recombineering is directed by sequence homology, not convenient restriction sites, it can be used to create genetic alterations on large DNA molecules such as BACs or chromosomes in a way not possible with classical in vitro genetic engineering techniques. Because of this, recombineering has expanded the analytical genetic capability of many organisms. Recombineering with ssDNA is over 100-fold more efficient than with dsDNA as a substrate. Using ssDNA it is possible to achieve recombination frequencies of over 50% of the cells that survive electroporation, thus a selection is not necessary for finding the desired mutations. The key to these high frequencies is avoiding the bacterial methyl-directed mismatch repair system, which normally removes greater than 99% of the incorporated changes. Avoiding mismatch repair can be easily achieved even in wild-type cells with careful oligonucleotide design.