Directed evolution of the site specificity of Cre recombinase

Abstract

Cre recombinase from bacteriophage P1 recognizes a 34-bp recombination site, loxP, with exquisite sequence specificity and catalyzes the site-specific insertion, excision, or rearrangement of DNA. To better understand the molecular basis of protein-DNA recognition and generate recombinases with altered specificities, we have developed a directed evolution strategy that can be used to identify recombinases that recognize variant loxP sites. To be selected, members of a library of Cre variants produced by targeted random mutagenesis must rapidly catalyze recombination, in vivo, between two variant loxP sites that are located on a reporter plasmid. Recombination results in an altered pattern of fluorescent protein expression that can be identified by flow cytometry. Fluorescence-activated cell sorting can be used either to screen positively for recombinase variants that recognize a novel loxP site, or negatively for variants that cannot recognize the wild-type loxP site. The use of positive screening alone resulted in a relaxation of recombination site specificity, whereas a combination of positive and negative screening resulted in a switching of specificity. One of the identified recombinases selectively recombines a novel recombination site and operates at a rate identical to that of wild-type Cre. Analysis of the sequences of the resulting Cre variants provides insight into the evolution of these altered specificities. This and other systems should contribute to our understanding of protein-DNA recognition and may eventually be used to evolve custom-tailored recombinases that can be used for gene study and inactivation.

Figure 1

LoxP, the natural recombination site of Cre. The outermost 13-bp regions on each side (black) are inverted repeats. The middle region (gray) is asymmetric and confers directionality to the site. The base pair identities most important for Cre binding (12) are boxed. Arrows indicate sites of cleavage during recombination.

Figure 2

Recombinase library and reporter system. (A) Plasmid system for genetic screening. The pR plasmid (Left) contains the Cre gene under control of the ara promoter (P_BAD), the araC gene, which allows modulated expression from P_BAD, a kanamycin selectable marker (kn^r), and a p15a origin of replication (ori). The pS plasmid (Right) contains, in its starting arrangement, two loxP sites (lox 1 and lox 2), the EYFP gene under control of the T7/lac promoter (P_T7), GFPuv downstream of a transcription termination region (rrnB ttr), the lacI gene, which allows inducible expression from P_T7, an ampicillin selectable marker (amp^r), and a colE1 origin of replication (ori). The EYFP and GFPuv genes are flanked by ribosome binding sites (RBS) and T7 RNA polymerase transcription termination regions (T7 ttr). Plasmids pS-M1–M8 have identical lox M1–M8 variant sites at lox 1 and lox 2. (B) Recombination at the lox 1 and lox 2 sites within pS and pS-M plasmids results in rearrangement of the plasmid and reorientation of P_T7 upstream of the GFPuv gene. Recombination is reversible. Cre recombination in vivo can be followed by fluorimetry (C) or cytometry (D) of cells. Cells containing pS alone (Left) express only EYFP, whereas cells containing pR and pS (Right) express roughly equal amounts of GFPuv and EYFP.

Figure 3

Cre recombination of loxP and loxP variant sites. Each of the eight loxP variants shown (M1–M8) were inserted into pS, replacing both of the loxP sites, and tested for recombination in vivo. All of the sites, except lox M3, M5, and M7, supported detectable levels of recombination by wild-type Cre.

Figure 4

Targeted Cre libraries. (A) The C1 library, in which residues M44, V48, T87, Q90, H91, and Y283 (red) have been randomized, was designed for use with the lox M5 site, in which base pairs 2 and 3 (green) have been altered. (B) The C2 library, in which residues I174, T258, R259, E262, and E266 (red) have been randomized, was designed for use with the lox M7 site, in which base pairs 5, 6, and 7 (green) have been altered.

Figure 5

In vivo activity of recombinase variants. Recombination activity is assessed as a ratio of EYFP (yellow) and GFPuv (green) expression, reflecting the relative concentrations of the two reporter plasmid arrangements in E. coli. Cells containing an active recombinase should contain approximately equal concentrations of the two reporter arrangements and express EYFP and GFPuv in roughly equal abundance.

Figure 6

Models of interactions of wild-type and C2 variant recombinases with loxP and lox M7. Interactions between Cre residues varied within the C2 library and base pairs 5–7 of loxP (Left) are based on crystal structural analysis (–6). Possible interactions between C2(+) #1 and lox M7 (Center) and C2(±) #4 and lox M7 (Right) are indicated by dashed lines.

Figure 7

In vitro activity of C2(±) #4 and wild-type Cre recombinases. (A) Schematic representation of the in vitro recombination reaction. Double-stranded DNA is shown in black; lox sites are shown as blue arrows. Recombination results in excision. (B) Enzyme concentration dependence of recombination. Reactions were carried out with in the presence of 0.1 nM substrate for 2 h. (C) Time courses of recombination. Agarose gels showing the substrate and one of the reaction products were ethidium bromide stained and analyzed by densitometry. Reactions were carried out in the presence of 0.5 nM substrate and 1000 nM enzyme.