The yeast assay for TALEN function was adapted from one we developed previously for ZFNs (8 (link),24 (link)) in which cleavage of the target, positioned between partially duplicated fragments of the lacZ gene, reconstitutes the gene via subsequent recombination to provide a quantitative readout (Supplementary Figure S3 a). For typical heterodimeric target sites (i.e. such as would typically occur in a native DNA sequence), paired TALEN constructs, in pTAL3 and pTAL4, are transformed together into yeast strain YPH500 (α mating type) using histidine and leucine prototrophy for selection. Individual TALEN monomers can be tested on homodimeric sites using just one of these plasmids. The target is made using synthesized complementary oligonucleotides that produce BglII- and SpeI-compatible ends and cloned between the lacZ fragments in the high copy DNA cleavage reporter plasmid pCP5 (24 (link)) cut with those enzymes (Supplementary Figure S3 b). The target plasmid is transformed into yeast strain YPH499 (α mating type), using tryptophan prototrophy for selection, but also excluding uracil from the growth medium: in addition to the target cloning site, pCP5 carries also the URA3 gene between the lacZ fragments so that selection for URA3 ensures that the strain has not undergone spontaneous recombination (and loss of URA3) prior to the assay.
Three transformants each of YPH500 carrying the TALEN construct(s) and of YPH499 carrying the target plasmid are cultured overnight at 30°C, with rotary shaking at 800 rpm, in synthetic complete medium lacking histidine and/or leucine (TALENs) or tryptophan and uracil (target). TALEN and target transformants are next mated (three pairs) by combining 200–500 µl of the overnight cultures, adding 1 ml of YPD medium and incubating for 4–6 h at 30°C, shaking at 250–300 rpm. Cells are harvested by centrifguation, washed in 1 ml synthetic complete medium lacking histidine and/or leucine and tryptophan, but now containing uracil, then resuspended in 5 ml of that medium and incubated overnight again at 30°C, with shaking (800 rpm), to an OD600 between 0.1 and 0.9. Cells are harvested by centrifugation, then resuspended and lysed using YeastBuster Protein Extraction Reagent (Novagen) according to the manufacturer's protocol for small cultures. A total of 100 µl of lysate is transferred to a microtiter well plate and β-galactosidase activity measured and normalized as previously described (24 (link)). For high-throughput, yeast may be cultured and mated (using a gas permeable seal) as well as lysed in 24-well blocks. We typically express activity relative to a Zif268 ZFN (24 (link)).
Three transformants each of YPH500 carrying the TALEN construct(s) and of YPH499 carrying the target plasmid are cultured overnight at 30°C, with rotary shaking at 800 rpm, in synthetic complete medium lacking histidine and/or leucine (TALENs) or tryptophan and uracil (target). TALEN and target transformants are next mated (three pairs) by combining 200–500 µl of the overnight cultures, adding 1 ml of YPD medium and incubating for 4–6 h at 30°C, shaking at 250–300 rpm. Cells are harvested by centrifguation, washed in 1 ml synthetic complete medium lacking histidine and/or leucine and tryptophan, but now containing uracil, then resuspended in 5 ml of that medium and incubated overnight again at 30°C, with shaking (800 rpm), to an OD600 between 0.1 and 0.9. Cells are harvested by centrifugation, then resuspended and lysed using YeastBuster Protein Extraction Reagent (Novagen) according to the manufacturer's protocol for small cultures. A total of 100 µl of lysate is transferred to a microtiter well plate and β-galactosidase activity measured and normalized as previously described (24 (link)). For high-throughput, yeast may be cultured and mated (using a gas permeable seal) as well as lysed in 24-well blocks. We typically express activity relative to a Zif268 ZFN (24 (link)).
