Mammalian genomes contain active recombinase recognition sites

Abstract

Recombinases derived from microorganisms mediate efficient site-specific recombination. For example, the Cre recombinase from bacteriophage P1 efficiently carries out recombination at its loxP target sites. While this enzyme can function in mammalian cells, the 34 bp loxP site is expected to be absent from mammalian genomes. We have discovered that sequences from the human and mouse genomes surprisingly divergent from loxP can support Cre-mediated recombination at up to 100% of the efficiency of the native loxP site in bacterial assays. Transient assays in human cells demonstrate that such pseudo-lox sites also support Cre-mediated integration and excision in the human cell environment. Pseudo sites for Cre and other recombinases may be useful for site-specific insertion of exogenous genes into mammalian genomes during gene therapy and other genetic engineering processes.

Introduction

Genetic engineering of mammalian cells often requires making permanent changes in the genome of target cells (Verma and Somia, 1997). An appealing strategy for permanence would involve site-specific integration of introduced sequences into safe positions in the genome. Homologous recombination can provide a high degree of specificity of position, but its efficiency is low (Vega, 1991). Recombination that is both highly specific and efficient can be mediated by recombinases isolated from microorganisms.

In some cases, recombinases have been shown to function in higher eukaryotic cells. For example, the Cre recombinase derived from E. coli phage P1 works well in mammalian cells (Sauer and Henderson, 1988, Sauer and Henderson, 1990, Sauer, 1994). Cre can mediate either excision or integration, depending on placement of its target loxP sites. The loxP recombination site is 34 bp long and consists of two 13 bp palindromes separated by an asymmetric 8 bp core sequence (Hoess et al., 1982). On a statistical basis, loxP is not expected to occur in mammalian genomes. To use Cre-mediated integration in mammalian cells, loxP sites have been first placed into the genome by imprecise and/or low-frequency procedures (Sauer, 1994). Recombinase-mediated integration would be more attractive for genetic engineering if sites that could act as targets for the Cre recombinase occurred endogenously in mammalian genomes at safe locations, such as regions outside of known genes.

It has been shown that most bases in the loxP core sequence can deviate from wild-type, as long as the cores match between the two recombining lox sites (Hoess et al., 1986). Minor changes in the loxP palindromes have also been shown to allow for Cre function in several cases (Abremski and Hoess, 1985, Abremski et al., 1988, Albert et al., 1995, Sauer, 1996, Sauer, 1992). Statistically, the presence of active lox sites in mammalian genomes requires that more significant departures from the loxP sequence be adequate to allow efficient Cre function. In the E. coli genome, a poor match to the loxP site called loxB occurs, but functions several orders of magnitude less well than loxP (Hoess et al., 1982, Sternberg et al., 1981). Similarly, in the yeast genome, several sites with a poor (10⁻⁵) function were identified (Sauer, 1992). Such sites would have little utility for genetic engineering because of their limited efficiency.

More recently, a single site in the yeast genome with approximately a third of the wild-type loxP function was identified (Sauer, 1996). This site has a remarkable degree of homology to loxP, including identity at 21 of 26 positions in the palindromes. It was not clear if the activity of the yeast site derived from fortuitous matching at important bases. On the hypothesis that a sufficiently small subset of the base pairs in the loxP palindromes may be adequate for function, and using genetic, biochemical, and structural data to identify relevant bases, we searched the human and mouse databases for pseudo-lox sites and tested candidate sites for function. We thus identified the first endogenous functional lox sites from higher eukaryotes.