7.16: Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP sites are around 34 basepairs in length. LoxP sites contain 13 bp palindromic sequences, meaning that the nucleotide sequence reads the same in both 5’ to 3’ and 3’ to 5’ directions. Site-specific recombination mediated by Cre recombinases is one of the most popular methods used in the creation of transgenic mice with acquired mutations. Using thermostable variants of Cre recombinase with tissue specific promoters allows for spatial control over Cre recombinase's expression and action. For instance, placing a kidney-specific Cadherin promoter upstream of the Cre gene allows the enzyme to be expressed only in renal tissues. For temporal control of Cre recombinase activity, the enzyme gene is fused with a ligand binding domain so that the enzyme is expressed only in the specific ligand’s presence.
A major limitation in using site-specific recombination as a genome editing tool is that the recombination target site or sites must be first inserted or must be present by chance. If a genomic site congruent with the enzyme recognition site can be preselected, the recombinases can be used with “made-to-order” target specificity. Recent studies have used mutagenesis and gene shuffling to design Flp variants that can functionally recognize sites with combinatorial mutations. The results are promising for future iterations of gene shuffling that can yield more specific Flp variants and can be used commercially as a molecular tool for engineering large genomes.
