Activity of the double-mutant proteins derived from the V2 or V3 mutants. (a) Cleavage activity in an extrachromosomal assay in CHO-K1 cells (described in Figure 1a). Each homodimer and derivative was tested against its cognate palindromic target. V2 and V3: expression of the V2 and V3 constructs, respectively. Mutations are indicated beneath the protein. LL and RR targets; are the palindromic sequences cleaved by the V2/V2 and V3/V3 homodimeric proteins, respectively. The mutants chosen for further analysis are noted V2⁺, V3⁻. Bars indicate the mean ± SD (n = 4). (b) Expression profile of V2 and V3 derived mutants in CHO-K1 cells. Transfected CHO-K1 cells were harvested after 48 h and lysates were probed with antibodies against the HA-Tag or the S-Tag. V2/V3, V2⁺/V3⁻; co-expression of the V2 and V3, and V2⁺ and V3⁻ mutant constructs respectively. V2, V2⁺, V3 and V3⁻; expression of V2, V2⁺, V3 and V3⁻, respectively. The results are expressed as a percentage of the non-mutated protein activity (c). Depletion of HA-tagged proteins from the supernatant. The lysate of transfected CHO-K1 cells was depleted of HA-tagged proteins by two successive immunoprecipitations. The successive supernatants, S initial (Si), S1 and S2 were probed by anti-HA and anti-S-Tag antibodies on western blots. V2/V3, V2⁺/V3⁻; co-expression of the V2 and V3, and V2⁺ and V3⁻ mutant constructs respectively. Antibody against β-actin was used for the loading control.

Gene targeting in a chromosomal reporter system in CHO-K1 cells. (a) Chromosomal assay used to monitor gene targeting induced by engineered meganucleases in CHO-K1 cells. Left panel: CHO-K1 cells containing a single-chromosomal copy of the lacZ gene interrupted by the DNA target site (black box) were co-transfected with meganuclease-expressing vectors and a repair plasmid. Target cleavage induces a gene targeting event that restores a functional β-galactosidase gene, by homologous recombination between the chromosomal reporter and the repair plasmid. We monitored the activity of the RAG1 meganucleases and of I-SceI, using two different cell lines, differing only in the meganuclease target site (LR for the RAG1 meganucleases, I-SceI target for the I-SceI meganuclease). Right panel: dose-response study. CHO-K1 cell lines were transfected with various amounts of expression vector for various meganucleases and a fixed quantity of the repair plasmid. (b) Meganuclease levels in CHO-K1 cells. Protein levels were monitored in CHO-K1 cells by western blotting, using the same amounts of plasmid as for the activity study. V2/V3, co-expression of the V2 and V3 constructs. scV3-V2(G19S), production of the single-chain protein. Protein levels were analyzed after transfection with various quantities of expression plasmids, from 10 to 1000 ng. Ø: empty vector.

Targeted integration at the endogenous RAG1 locus, driven by a single-chain meganuclease. (a) Experimental outline and diagram of the gene targeting strategy used at the endogenous RAG1 locus. The RAG1 target sequence is located just upstream from the coding sequence for exon 2 of the Rag1 protein. Exon 2 is boxed, with the open reading frame in white. Cleavage of the native RAG1 gene by the meganuclease yields a substrate for homologous recombination, which may use the repair plasmid containing either 10 bp or 1.7 kb of exogenous DNA. The 10-bp DNA fragment (containing a single HindIII restriction site) is flanked by two sequences, 900 bp and 500 bp in length, homologous to the human RAG1 locus. The 1700 bp DNA fragment is flanked by two homology arms of 2.0 kb and 1.6 kb in length. Human 293H cells were transfected with 3 µg of scV3⁻-V2⁺(G19S) meganuclease expression plasmid and 2 µg of the repair substrate, cultured without selection for 48 h, re-plated, and individual clones picked and amplified. Targeted integration events were detected by PCR amplification and subsequently confirmed by Southern blotting. (b) Example of a screen for targeted integration events with a repair plasmid containing 10 bp of exogenous DNA. Genomic DNA derived from individual clones (a–i) obtained after transfection with a meganuclease expression plasmid and a repair plasmid was amplified by PCR and digested with HindIII. − is the negative control (non-transfected cells). PCR primers positions are indicated by simple arrows, with one primer binding outside the region of homology used in the repair plasmid. On the gel, bands correlated with a targeted event are indicated with a black triangle (c) Example of a PCR screen for targeted integration events with the repair plasmid containing 1700 bp of exogenous DNA. PCR analysis of 11 different samples (1–11), + indicates positive control (a recombinant plasmid), and − is the negative control (non-transfected cells). (d) Southern blot analysis of six individual clones (1–6) that were positive in the PCR screen for the integration of the 10 bp exogenous fragment, analyzed together with DNA from non-transfected cells (−). The locus maps indicate the restriction pattern of the wild-type locus (5.3 kb) and the targeted locus (3.0 kb). (e) Southern blot analysis of three PCR-positive (1–3) and two PCR-negative clones (4–5) samples derived from single transfected cells are analyzed, together with DNA from non-transfected cells (−). The locus maps indicate the restriction pattern of the wild-type locus (5.3 kb) and the targeted locus (3.4 kb). Probes are indicated by a solid black box. H, genomic location of HindIII cleavage sites.

Design of obligate heterodimers and single-chain molecules. (a) Extrachromosomal assay used to monitor the activity of the engineered meganucleases in CHO-K1 cells. A meganuclease cleavage site (black box) is placed between two direct repeats in a reporter plasmid. Cleavage of the target site induces tandem-repeat recombination, thereby restoring a functional β-galactosidase gene. V2/V3: co-expression of the V2 and V3 mutant constructs; V2 and V3: expression of V2 and V3, respectively; Ø: empty vector. Cleavage activity was monitored against the palindromic targets LL (gray bars) and RR (white bars), and the non-palindromic target LR (black bars). The cleavage activity of I-SceI with its target is shown as a positive control (hatched bar). Bars indicate the mean ± SD (n = 3). (b) Engineered positions are indicated on the structure of the I-CreI meganuclease bound to its DNA substrate (PDB code 1G9Y). The three monomer–monomer interactions targeted in the obligate heterodimer design are highlighted. In addition, the position of the two Gly19 residues is indicated by two blue spheres. C₁ and N₂ indicate the N- and C-termini of the first and second monomer, respectively. The dashed gray line represents a linker used to generate single-chain molecules.

Activity of the obligate heterodimers and single-chain meganucleases (a) Extrachromosomal assay in CHO-K1 cells. The principle of the assay is described in Figure 1a. Cells were transfected with meganuclease constructs of various designs and the resulting cleavage of the three targets LR (black bars), LL (gray bars) and RR (white bars) is shown as a percentage of the activity resulting from V2/V3 co-expression. V2+, mutant V2 (E8K, E61R); V3−, mutant V3 (K7E, K96E); sc, single-chain molecule; Ø: empty vector. Bars indicate the mean ± SD (n = 3). (b) In vitro cleavage activity of heterodimers and single-chain molecules against the LR target. The reaction mixture included a target concentration of 2 nM and the purified protein. Gels from the left panel were quantified by densitometry. The graph in the right panel summarizes the data. Lanes 1–15; protein concentrations of 120, 90, 60, 40, 30, 20, 10, 7.5, 5, 3.5, 2, 1, 0.5, 0.25 and 0 nM, respectively. C₅₀; enzyme concentration required to cleave 50% of the target DNA.

Evaluation of the toxicity of meganucleases. (a) Toxicity of the engineered meganucleases, as monitored by a cell survival assay. Various amounts of meganuclease expression vector and a constant amount of plasmid encoding GFP were used to co-transfect CHO-K1 cells. Cell survival is expressed as the percentage of cells expressing GFP 6 days after transfection, as described in the ‘Materials and Methods’ section. The inactive V2(G19S)/V3(G19S) heterodimer is shown as a control for non-toxicity. (b) DNA damage was visualized by the formation of γ-H2AX foci at DNA double-strand breaks. CHO-K1 cells were transfected with a plasmid encoding the meganuclease fused to an HA epitope at the active dose (quantity used for maximal activity) or 10 times the active dose. The inactive V2(G19S)/V3(G19S) heterodimer was used as a control (1 µg of each expression plasmid). The number of γ-H2AX foci in transfected cells (HA positive) was determined for each condition. Representative images below each class illustrate the different classes of γ-H2AX foci in CHO-K1 cells transfected with meganuclease: Red, HA labeling; green, γ-H2AX foci; and blue, DAPI staining.