A method for performing precise alterations in the yeast genome using a recycable selectable marker.

A new strategy has recently been described for performing direct gene deletion in Saccharomyces cerevisiae, based on the use of a PCR-amplified DNA fragment which includes a selectable marker flanked by two sequences of -35-50 base pairs (bp), respectively, homologous to two chosen targets in the genome (1). The method is extremely useful since it does not require the construction of disrupted genes, but has, however, two main limitations. First, as S.cerevisiae (Sc) genes are used as selectable markers, it is necessary to work with recipient strains in which these genes are completely deleted, in order to avoid gene conversions. Indeed, such events are frequent compared to the desired recombinations, since the lengths of the two sequences homologous to the target gene are very short. In addition, after deletion of the gene the selectable marker remains integrated in the genome and can no longer be used in the modified strain. Other markers must therefore be available for performing additional gene disruptions. In this work a method was adapted for circumventing these two limitations, and was also shown to be suitable for creating site-specific genomic mutations in addition to deletions. The Kluyveromyces lactis (Kl) URA3 gene was used as selectable marker in order to minimize non-desired recombination events with ura3 genomic sequences, as these genes are significantly divergent (-68% identical nt, -81% identical aa). Comparison of site-specific integration efficiencies obtained using Kl.URA3 or Sc. URA3 was performed in the ura3-52 strain YPH500 according to the method described by Baudin and co-authors (1), with terminal sequences homologous to PMS1 (from nt 418 to 447 and from nt 2740 to 2769, databank M29688). With the Kl marker, the expected PMS1 deletion was obtained in 10 out of 12 clones analysed while no clone among 17 analysed candidates was found to carry the Sc marker at the PMS1 locus. This demonstrated the usefulness of utilizing the heterologous gene. Since a recyclable marker (2) was to be included in the system, the use of URA3 was convenient, as URAclones can be selected in the presence of fluoroorotic acid (FOA) (3). This choice was in fact critical in the specific context of the method developed here, as shown below. In previous gene replacement experiments, using a template consisting of URA3 surrounded by direct repeats of heterologous sequences of 250 bp, linear molecules with terminal homologies to a genomic target were produced by PCR, and site-specific integration as well as subsequent marker excision were efficiently selected. However, such a procedure was not applicable for targeting another region of the genome, using the same template. Indeed, preferential integration then occured by homologous recombination within the resident heterologous sequence that remained after URA3 excision at the first locus targeted (data not shown). In addition, such a system was not appropriate for performing site-specific mutagenesis. These problems were solved by creating terminal repeats of 50-60 bp including exclusively sequences from each genomic target by appropriate design of the synthetic oligonucleotides. The strategy permitted a compromise between the need for sufficient sequence length for the selection of excision events (selection of marker excision failed using 25 bp direct repeats surrounding URA3, data not shown) and the need for minimizing the size of the synthetic oligonucleotides (in the defined conditions, -80 nt long oligonucleotides were used). The two-step procedure for performing site-specific mutagenesis is outlined in Figure 1 and mutagenesis of TEC1 is described. Linear DNA molecules including the Kl URA3 gene were generated by PCR, using synthetic oligonucleotide primers with 5' extentions homologous to the genomic target sequence. A single bp modification leading to the creation ofa NcoI restriction site, was introduced in the middle of the 50 bp repeats. After transformation of a ura3 strain (W303-1B) with this PCR fragment, URA+ colonies were selected on minimal medium, purified, and the targeted genomic locus was analysed (Fig. 1). Two out of nine clones appeared to carry URA3 integrated within TEC1 together with two mutated direct repeats. The others were shown to carry unmodified TEC1 locus by similar analysis. In these latter cases, integration presumably occured at another locus (possibly ura3). A clone in which site-specific integration of URA3 together with the 50 bp flanking sequences took place was then streaked on non-selective YEPD medium. After 48 h growth at 30°C, about 107 cells were resuspended in water and plated on FOA selective medium. Isolated colonies were purified on the same FOA solid medium. The genomic locus was then analysed in order to identify the ura3 clones of interest (Fig. 1). In four out of 11 clones, the precise URA3 excision resulting from homologous recombination between the 50 bp direct repeats, giving rise to the desired genomic point mutation, was shown to have taken place. Concerning the other candidates, spontaneous genomic mutations were presumably responsible for the FOA resistance. A similar strategy was also adapted for creating precise genomic deletions. The procedure is outlined in Figure 2 and deletion ofPMS1 in YPH500 is described as an example. Linear DNA molecules including the Ki URA3 gene were synthesised by PCR using synthetic oligonucleotide primers with 5' extensions