Construction of target clones
The 22-bp LR target found in the human RAG1 gene (TGTTCTCAGGTACCTCAGCCAG), the two palindromic derivative targets LL (TGTTCTCAGGTACCTGAGAACA) and RR (CTGGCTGAGGTACCTCAGCCAG) were cloned as follows: oligonucleotides containing the target site (Proligo) were amplified by PCR to generate double-stranded target DNA and then inserted into reporter vectors with the Gateway protocol (Invitrogen): the yeast vector pFL39-ADH-LACURAZ and the mammalian vector pcDNA3.1-LAACZ, both previously described and containing an I-SceI target site as a control. Yeast reporter vectors were used to transform Saccharomyces cerevisiae strain FYBL2-7B (MAT a, ura3Δ851, trp1Δ63, leu2Δ1, lys2Δ202).
Construction of the single-chain molecule
One single-chain molecule contains two almost identical sequences, encoding the I-CreI derived V2 and V3 mutants. The two coding sequences are different, displaying only 72% nucleic acid sequence identity, thereby avoiding problems relating to construct stability. The N-terminal half of the single-chain molecules contains a full length I-CreI molecule of 163 residues, whereas the C-terminal sequence lacks the first five residues. Various linkers were used to connect the two mutant proteins ().
Amino acid sequences of the linker regions that were inserted between two I-CreI-variants V2 and V3
The ORFs encoding the various meganucleases were amplified by PCR and inserted into the 2 μm-based replicative vector pCLS542, harboring the LEU2 gene. Saccharomyces cerevisiae strain FYC2-6A (MATα, trp1Δ63, leu2Δ1, his3Δ200) was then transformed with the vector using a high-efficiency lithium acetate transformation protocol. The various meganuclease genes were also inserted into the mammalian vector pcDNA3.1. The genes were expressed under the control of a CMV promoter. Protein production in CHO-K1 cells was monitored by adding an HA (YPYDVPDYA) or a S-Tag (KETAAAKFERQHMDS) epitope to the C-terminus of the protein. Cells were harvested 48 h after the transfection of the cells using the Polyfect reagent (Qiagen) and directly solubilized in Laemmli buffer (100 µl of buffer for 106 cells). The equivalent of 105 cells was loaded on a SDS–PAGE gel and probed by western blot using an anti-HA antibody (Roche) or an anti-S-Tag antibody (Santa Cruz).
Purification of meganucleases
For the production of heterodimeric I-CreI derivatives, the ORFs of each of the monomers were inserted into the CDFDuet-1 vector (Novagen) with a 6xHis tag or a Strep tag at the C-terminus. The double-tagged heterodimers were produced and purified as previously described (30
). The protein was concentrated to about 15 mg/ml, flash-frozen in liquid nitrogen and stored at −80°C. Protein concentration was determined by measuring absorbance at 280 nm. Sample purity was checked by SDS–PAGE and heterodimer formation was assessed by western blotting with an anti-His or an anti-Strep tag antibody and confirmed by mass spectrometry. All the purified proteins were found to have adopted a structure similar to that of the wild type (as shown by circular dichroism and NMR) and to be dimeric in solution (as shown by analytical ultracentrifugation: data not shown).
The sequence encoding the single-chain scV3-V2(G19S) protein with a 6xHis tag at the C-terminus was inserted into the pET24d(+) vector and expressed in Escherichia coli Rosetta(DE3)pLysS cells (Novagen) grown in LB supplemented with kanamycin and chloramphenicol. Induction with IPTG for 5 h at 37°C or for 15 h at 20°C yielded high levels of expression, however, after sonication in lysis buffer containing 50 mM sodium phosphate pH 8.0, 300 mM NaCl, 5% glycerol and protease inhibitors (Complete EDTA-Free tablets, Roche) and ultracentrifugation at 20 000 × g for 1 h, the protein was found exclusively in the insoluble fraction as determined by western blots with an anti-His antibody. The protein was therefore purified under denaturing conditions by first solubilizing it in lysis buffer supplemented with 8 M urea. After clarification by ultracentrifugation (2 h at 40 000 × g) the sample was applied onto a column packed with Q-Sepharose XL resin (GE Healthcare) equilibrated with the same buffer. This purification step separated all the nucleic acids (retained in the column) from the protein and improved the performance of subsequent purification steps. The protein was recovered from the flow through on a Co2+-loaded HiTrap Chelating HP 5 ml column (GE Healthcare) equilibrated with the lysis buffer plus 8 M urea. After sample loading and column washing the protein was eluted with the same buffer supplemented with 0.5 M imidazol. Protein-rich fractions (as determined by SDS–PAGE) were collected and allowed to undergo refolding, through a 20-fold dilution (drop by drop) with 20 mM sodium phosphate pH 6.0, 300 mM NaCl at 4°C (final protein concentration of 0.13 mg/ml). The refolded protein was loaded onto a 5 ml HiTrap heparin column equilibrated with the same buffer and eluted with a gradient to 1 M NaCl. The fractions containing the pure protein were pooled, concentrated up to 1.4 mg/ml (35.6 μM, determined by absorbance at 280 nm) and were either used immediately or flash frozen in liquid nitrogen and stored at −80°C. The identity of the protein was confirmed by mass spectrometry which showed that the initial methionine was absent from the purified polypeptide chain. The purified protein was found to be folded into a structure similar to that of the wild type by circular dichroism and NMR (data not shown), and to be monomeric in solution by analytical gel filtration.
In vitro cleavage assay conditions
Cleavage assays were performed at 37°C in 10 mM Tris–HCl pH 8, 50 mM NaCl, 10 mM MgCl2
. The target concentration was 2 nM (target substrates in plasmid pGEM-T linearized with XmnI) and the protein concentrations were 120 (for cleavage detection) and 120, 90, 60, 40, 30, 20, 10, 7.5, 5, 3.5, 2, 1, 0.5, 0.25 and 0 nM for the quantification of cleavage efficiency, in a final reaction volume of 25–50 μl. Reactions were stopped after 1 h, adding 5 μl of 45% glycerol, 95 mM EDTA (pH 8), 1.5% (w/v) SDS, 1.5 mg/ml proteinase K and 0.048% (w/v) bromophenol blue (6× stop buffer) and incubating at 37°C for 30 min. The products of the reaction were subjected to electrophoresis in a 1% agarose gel. The gels were stained with SYBR Safe DNA gel staining kits (Invitrogen) and the intensity of the bands observed upon illumination with UV light was determined with the ImageJ software (http://rsb.info.nih.gov/ij/
). The linearized target plasmid was 3 kb in size and yielded two smaller bands, of 2 kb and 1 kb, upon cleavage with the meganuclease. The percentage of cleavage was calculated with the following equation: % cleavage = 100 × (I2 kb
+ I1 kb
+ I2 kb
+ I1 kb
), where I1 kb
, I2 kb
and I3 kb
are the intensities of the 1, 2 and 3 kb bands, respectively.
Mating of meganuclease expressing clones and screening in yeast
A colony gridder (QpixII, Genetix) was used for the mating of yeast strains. Mutants were gridded on nylon filters covering YPD plates, using a high gridding density (about 20 spots/cm2). A second gridding process was performed on the same filters for the spotting of a second layer consisting of reporter-harboring yeast strains for each target. Membranes were placed on solid agar YPD rich medium, and incubated overnight at 30°C, to allow mating. The filters were then transferred onto synthetic medium, lacking leucine and tryptophan, with galactose (2%) as the carbon source (and with G418 for co-expression experiments), and incubated for 5 days at 37°C, to select for diploids carrying the expression and target vectors. Filters were then placed on solid agarose medium with 0.02% X-Gal in 0.5 M sodium phosphate buffer, pH 7.0, 0.1% SDS, 6% dimethyl formamide (DMF), 7 mM β-mercaptoethanol, 1% agarose, and incubated at 37°C, to monitor β-galactosidase activity. Filters were scanned and quantified with proprietary software. Beta-galactosidase activity is directly associated with the efficiency of homologous recombination. Experiments using several purified I-CreI mutants with various recombination activities in yeast have shown that the recombination efficiency quantified in yeast is directly correlated with the cleavage activity in vitro (unpublished data).
Extrachromosomal assay in CHO-K1 cells
CHO-K1 cells were transfected with the meganuclease expression vectors and the reporter plasmid, in the presence of Polyfect transfection reagent in accordance with the manufacturer's protocol (Qiagen) The culture medium was removed 72 h after transfection, and 150 μl of lysis/detection buffer was added for β-galactosidase liquid assay (typically, for 1 l of buffer, we used 100 ml of lysis buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Triton X100, 0.1 mg/ml BSA, protease inhibitors), 10 ml of Mg 100× buffer (MgCl2 100 mM, 2-mercaptoethanol 35%), 110 ml of an 8 mg/ml solution of ONPG and 780 ml of 0.1 M sodium phosphate pH 7.5). After incubation at 37°C, we measured optical density at 420 nm. The entire process was performed on an automated Velocity11 BioCel platform.
CHO-K1 cells were transfected with the meganuclease expression vectors in the presence of Polyfect transfection reagent according to the manufacturer's (Qiagen) protocol. Cells were harvested 48 h after transfection. We avoided the disruption of heterodimers due to the use of stringent buffers such RIPA, by resuspending cells in PBS supplemented with a mixture of protease inhibitors (Santa Cruz) and lysing them by successive freeze-thaw cycles. After centrifugation (10 min at 13 000 × g), the supernatant was recovered and the total protein concentration was determined by the BCA assay. Volumes were adjusted such that the protein concentration was identical (0.7 mg/ml) for the V2/V3 heterodimer and the V2+/V3− obligate heterodimer. Both supernatants were then incubated twice, for 12 h each, with 15 µl (equivalent to 7.5 µg of antibody) of an anti-HA antibody coupled to agarose at 4°C. The sample was then centrifuged (2 min at 8000 × g) and 10 µl of the supernatant were subjected to SDS–PAGE and western blotting.
Targeting of a chromosomal reporter gene in CHO-K1 cells
CHO-K1 cell lines harbouring the reporter system were seeded at a density of 2 × 105 cells per 10 cm dish in complete medium [Kaighn's modified F-12 medium (F12-K), supplemented with 2 mM l-glutamine, penicillin (100 IU/ml), streptomycin (100 µg/ml), amphotericin B (Fongizone) (0.25 µg/ml) (Invitrogen-Life Science) and 10% FBS (Sigma-Aldrich Chimie)]. The next day, cells were transfected in the presence of Polyfect transfection reagent (Qiagen). Cells were co-transfected with 2 µg of LacZ repair matrix vector and various amounts of meganuclease expression vector. After 72 h of incubation at 37°C, cells were fixed by incubation in 0.5% glutaraldehyde at 4°C for 10 min, washed twice in 100 mM phosphate buffer supplemented with 0.02% NP40 and stained with the following staining buffer [10 mM phosphate buffer, 1 mM MgCl2, 33 mM potassium hexacyanoferrate (III), 33 mM potassium hexacyanoferrate (II), 0.1% (w/v) X-Gal]. Plates were incubated overnight at 37°C and examined under a light microscope to determine the number of LacZ-positive cells. The frequency of LacZ repair is expressed as a percentage and is calculated as the number of LacZ-positive foci divided by the number of transfected cells (5 × 105), corrected for the transfection efficiency.
Cell survival assay
The CHO-K1 cell line was used to seed plates at a density of 2 × 105 cells per 10 cm dish. The next day, various amounts of meganuclease expression vectors and a constant amount of GFP-encoding plasmid were used to transfect the cells. GFP levels were monitored on Days 1 and 6 after transfection, by flow cytometry (Guava EasyCyte, Guava Technologies). Cell survival is expressed as a percentage and was calculated as a ratio: (meganuclease-transfected cell expressing GFP on Day 6/control transfected cell expressing GFP on Day 6), corrected for the transfection efficiency determined on Day 1.
For γ-H2AX immunocytochemistry, CHO-K1 cells were transfected with a mixture containing various amounts of plasmid encoding a HA-tagged meganuclease, with DNA levels made up to 4 µg with empty vector, in the presence of Polyfect reagent (Qiagen). Cells were fixed 48 h after transfection, by incubation with 2% of paraformaldehyde for 30 min, and permeabilized by incubation for 5 min at room temperature in 0.5% Triton. Cells were washed and incubated for 1 h in 0.3% Triton buffer supplemented with 10% normal goat serum (NGS) and 3% BSA, to block non-specific staining. Cells were then incubated for 1 h at RT with anti-γ-H2AX (Upstate: 1/10 000) and anti-HA (Santa Cruz:1/200) antibodies diluted in Triton 0.3% in PBS supplemented with 3% BSA and 10% NGS and then for 1 h with Alexa Fluor 488 goat anti-mouse (Invitrogen-Molecular Probes: 1/1000) and Alexa Fluor 546 goat anti-rabbit secondary antibodies diluted in 0.3% Triton in PBS supplemented with 3% BSA, and 10% NGS. Coverslips were incubated with 1 µg/ml 4′,6-diamino-2′-phenylindole (DAPI; Sigma), mounted and the γ-H2AX foci were visualized in transfected cells (HA-positive) by fluorescence microscopy. The inactive double-mutant V2(G19S)/V3(G19S) heterodimer was used as a negative control at a dose of 1 µg of each expression plasmid.
Endogenous gene targeting experiments
The donor plasmid for gene targeting experiments contained left and right homology arms generated by PCR amplification of the human RAG1 locus. An exogenous DNA fragment was inserted between these two arms. These sequences consisted of either a 1.7-kb DNA fragment derived from a neomycin expression plasmid or a 10-bp DNA sequence containing an HindIII recognition site. The human 293H cells (Invitrogen) were plated at a density of 1 × 106 cells per 10 cm dish in complete medium (DMEM supplemented with 2 mM l-glutamine, penicillin (100 IU/ml), streptomycin (100 µg/ml), amphotericin B (Fongizone: 0.25 µg/ml, Invitrogen-Life Science) and 10% FBS). The next day, cells were transfected in the presence of Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's protocol. Cells were co-transfected with 2 µg of the donor plasmid and 3 µg of meganuclease expression vector. After 48 h of incubation at 37°C, cells were treated with trypsin and dispensed at a density of 10 or 100 cells per well in 96-well plates or re-plated and individual clones picked and subsequently amplified. DNA was extracted with the ZR-96 genomic DNA kit (Zymo research) according to the manufacturer's protocol. PCR amplification reactions were performed with the primers F2:5′-AGGATCTCCTGTCATCTCAC-3′ and R2: 5′-GCAGTGTTGCAGATGTCACAG-3′, and F2:5′-AGGATCTCCTGTCATCTCAC-3′ and R12: 5′-CTTTCACAGTCCTGTACATCTTGT-3′ in order to detect the targeted integrations of the 10 bp and 1700 bp exogenous fragments, respectively. The PCR products related to the 10 bp integrations experiments were further digested by the restriction enzyme HindIII. Southern blot analysis was performed with genomic DNA digested with HindIII and hybridized with an 830 bp RAG1 specific probe binding outside of the right homology arm of the donor plasmid.