Size of gene specific inverted repeat--dependent gene deletion In Saccharomyces cerevisiae

Abstract

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted repeats of the URA3, which requires the engineering of unique restriction sites for subsequent digestion and T4 DNA ligation in vitro. We further identified that the excision of the entire mutagenesis cassette flanked by two DRs in the transformed S. cerevisiae is dependent on the length of the inverted repeat of which a minimum of 800 bp is required for effective gene deletion. The deletion efficiency improves with the increase of the inverted repeat till 1.2 kb. Finally, the use of gene-specific inverted repeats of target genes enables simultaneous gene deletions. The procedure has the potential for application on other yeast strains to achieve precise and efficient removal of gene sequences.

Figure 1. Scheme of the approach for gene deletion in S. cerevisiae using mutagenesis cassettes containing gene-specific inverted repeats.

A: Preparation of disruption gene cassette by OE-PCR. N1 through N5 are oligos for amplification of individual DNA fragments and the final disruption gene cassette. B: The procedure for rapid and scar-free gene deletion. The disruption cassette consisting of the URA3 marker and one copy of the inverted DNA sequence of a target gene is created by OE-PCR and flanked by 36 bp homologous arms. C: Deletion of NDE1 gene by the described approach in this study. CTR: parental S. cerevisiae strain; IR: inverted repeat; DR: direct repeat; GOI: gene of interest.

Figure 2. An illustration of simultaneous gene deletions in S. cerevisiae.

A: Preparation of disruption gene cassettes by utilizing prototrophy for the nucleotide uracil and tryptophan and counter-selection for their respective auxotrophs using 5′-FOA and 5-FFA. B: Initial confirmation of the integration of the disruption gene cassettes TRP1-irNDE1 and URA3-irNDE2 into NDE1 and NDE2 loci, respectively for simultaneous gene deletion with C1 and C2 confirmation oligos and genomic DNA isolates. C: PCR-confirmation of excision of the disruption gene cassettes post counter-selection using C1 and C4 oligo pair. CTR: parental S. cerevisiae strain; IR: inverted repeat; DR: directed repeat; GOI: gene of interest.