Evolution of variants of yeast site-specific recombinase Flp that utilize native genomic sequences as recombination target sites

Abstract

As a tool in directed genome manipulations, site-specific recombination is a double-edged sword. Exquisite specificity, while highly desirable, makes it imperative that the target site be first inserted at the desired genomic locale before it can be manipulated. We describe a combination of computational and experimental strategies, based on the tyrosine recombinase Flp and its target site FRT, to overcome this impediment. We document the systematic evolution of Flp variants that can utilize, in a bacterial assay, two sites from the human interleukin 10 gene, IL10, as recombination substrates. Recombination competence on an end target site is acquired via chimeric sites containing mixed sequences from FRT and the genomic locus. This is the first time that a tyrosine site-specific recombinase has been coaxed successfully to perform DNA exchange within naturally occurring sequences derived from a foreign genomic context. We demonstrate the ability of an Flp variant to mediate integration of a reporter cassette in Escherichia coli via recombination at one of the IL10-derived sites.

Figure 1

Contacts between Flp and its target site, FRT. In the FRT sequence, the 13 bp Flp-binding elements flanking the 8 bp spacer are indicated by the horizontal arrows. The individual bp within a binding element are shown in green upper case letters, and numbered in ascending order from the position nearest to the spacer. The spacer sequence is written in black lower case letters, and the scissile phosphates are indicated by ‘p’. (A and B) A subset of the contacts between the Flp protein and FRT relevant to the directed evolution of new target specificity are displayed (11).

Figure 2

FRT, FRT-like (FL) genomic sites and FLRT chimeric sites. (A) The FRT site is represented as in Figure 1. (B) The FRT-like (FL) sequences from the human IL10 gene used for this study were FL-IL10A (GenBank accession no. X78437, positions 1154–1187) and FL-IL10B (GenBank accession no. AF418271, positions 1264–1297). The positions within the FL-‘binding elements’ that differ from FRT are denoted by lower case red letters. TargetFinder scores for these sites, normalized to a score of 100 for FRT, are shown at the right. (C) In the chimeric sites between FRT and FL-IL10B, the spacer as well as positions 1–8 (FLRTB(1-8g); score 73) or positions 1–10 (FLRTB(1-10g); score 67) were derived from IL10B.

Figure 3

Evolution of Flp variants that show recombination competence on FLRTB(1-8g). The deletion reaction that converts the reporter plasmid P to plasmid R, and forms the basis of the colony color assay, is schematically depicted at the top. (A) The template for directed evolution was a variant Flp^sup3 described previously (26) that showed relaxed specificity towards FRT and three altered target sites derived from it. The set of mutations present in Flp^sup3 are listed (26). The subset of the sup3 mutations that were retained in the Flp variants that recombine FLRTB(1-8g) is denoted by sup3* (A35T, I45V, T50A, E150G, K173R, I295F, T324I, I367L). The recombination activity of a variant in the negative system (deletion of lacZα) was expressed as the percentage of white colonies on X-gal plates. Plasmids isolated from randomly picked white colonies (w) were digested with SphI to verify authentic recombination. Representative results in lanes 2–5 correspond to FV1–FV4, respectively. Lane 1 shows the digestion result for a blue colony (b) from the FV1 assay. P refers to the parent plasmid, and R to the larger of the two deletion products. The top band (EP) corresponds to the Flp (or Flp variant) expression plasmid. (B) The molar ratios of protein to binding element in the in vitro binding reactions of FV3 to FRT and FLRTB(1-8g) were ∼2, 4 and 6 (lanes 2–4). The bound complexes BC contain one FV3 monomer (lower band) or two monomers (upper band) per substrate molecule. The plot shows the levels of binding of FV3 to the two targets. Closed circle, FRT; open circle, FLRTB(1-8g). (C) In vitro recombination activity of FV3 was assayed on FRT and FLRTB(1-8g) using 2, 4 and 8 monomers of FV3 per binding element (lanes 2–4). P and R stand for parental and recombined plasmids. The small deletion circle that runs towards the bottom of the gel during electrophoresis is not shown.

Figure 4

Evolved variants of Flp with recombination activity on FLRTB(1-10g), FL-IL10A and Fl-IL10B. The recombination event in the parent plasmid P that excises the rrnB transcription terminator to cause lacZ expression from plasmid R is schematically shown at the top. (A) The mutations, in addition to the sup3* set (see legend to Figure 3), present in Flp variants that act on FLRTB(1-10g) are tabulated. Recombination assays were done using the positive system (expression of lacZα), and the activity of each variant was expressed as the percent of blue colonies on X-gal plates. Plasmids isolated from a random set of blue colonies (b) were digested with SphI to verify the predicted deletion event. Typical results for FV5–FV7 are shown in lanes 2–4, respectively. The result for a white colony (w) obtained with FV7 is shown in lane 1. P and R refer to the parental plasmid and the larger of the two deletion products, respectively. The band at the top denotes the expression plasmid (EP). The extra band above P in lanes 2–4 was either the product of the excision reaction from a plasmid dimer or resulted from insertion of the excised rrnB circle into a parental monomer plasmid. (B) Recombination efficiencies of Flp, Flp^sup3, FV7 and FV8 on the genomic sites FL-IL10A and FL-IL10B, as well as the authenticity of recombination, were assayed as described under (A). Plasmids isolated from blue (b) or white (w) colonies were digested with SphI. Only the results for FV7 are shown. The reporter substrates are indicated above the respective lanes. Recombination on FL-IL10B was quite weak but detectable. (C) Plasmid mixtures corresponding to lanes 3 and 4 of (A) were digested with SphI plus HindIII (lanes 3 and 4), and fractionated with similarly digested Flp expression plasmid (EP) (lane 1) and the parent reporter plasmid (P). The bands marked P1 and P2 refer to the backbone of the recombined reporter and the rrnB cassette, respectively. The size of the band marked ‘X’ agrees with it containing two copies of rrnB and three copies of the recombination target site. This is the expected configuration for an intermolecular recombination event that inserts a copy of the excised rrnB circle into the parent reporter plasmid. The lane containing the DNA size ladder is marked ‘M’.

Figure 5

Recombination between a genomic site and a partner FRT site or a hybrid site containing one Flp-binding element from FRT. (A and B) Recombination was assayed in the positive screen using the indicated proteins and the schematically diagrammed reporter constructs. The recombinase-binding elements are represented by colored arrows. Recombination events for FV7 were ascertained by SphI digestion of plasmids from blue colonies (b). Lanes 2–4 represent IL10B2, IL10B3 and IL10A3 reporter cassettes, respectively. Lane 1 shows the control digestion of plasmids from a white colony obtained in the assay with the IL10A3 reporter. (C) The in vitro recombination reactions were carried out using the reporter plasmid carrying the IL10A3 recombination cassette depicted in (B). The reactions in lanes 2 and 3 contained ∼8 monomers of Flp and FV7, respectively, per binding element. P and R refer to the parent plasmid and the larger of the two deletion products. The smaller deletion circle, which runs towards the bottom of the gel, is not shown. The band just above P in lane 3 is due to an intermolecular recombination event between the smaller deletion circle and P.

Figure 6

Site-specific integration at the IL10A site promoted by FV7. (A) The expected site-specific recombination event between pLac and pRFP plasmids is outlined. In the recombinant fusion plasmid pLac-RFP, lacZα would be positioned downstream of the Lac promoter (Pr). (B) Upon induction of FV7, expression of lacZα was observed in ∼45% of the colonies (blue on X-gal plates). (C) Digestion of plasmids from randomly picked white (w) and blue (b) colonies with XhoI confirmed the integration event.