In Saccharomyces cerevisiae gene targeting fidelity depends on a transformation method and proportion of the overall length of the transforming and targeted DNA

Abstract

Gene replacement is one of the most essential approaches in construction of the genetically modified yeast strains. However, the fidelity of gene targeting and the effort needed for construction of a particular strain can vary significantly. We investigated the influence of two important factors-the choice of the transformation method and the design of the transforming DNA fragment, which can vary in overall length (including flanking regions and selectable marker) compared to the length of the targeted region in the genome. Gene replacement fidelity was determined in several assays using electroporation and spheroplast transformation, and compared with our previous results obtained by lithium acetate. We have demonstrated clearly that gene targeting fidelity depends on the transformation protocol, being highest for lithium acetate method. In contrast, lower fidelity was observed with electroporation and spheroplast transformation. Additionally, the fidelity also depends on a design of the transformation assay, since a higher overall length ratio of the transforming DNA and targeted region results in higher fidelity. Moreover, the karyotype analysis of the aberrant transformants by qPCR demonstrates that gene targeting can result in diploidisation of haploid strains, most likely via targeted chromosome duplication followed by subsequent duplication of other chromosomes.

Development of a marker-free mutagenesis system using CRISPR-Cas9 in the pathogenic mould Aspergillus fumigatus

“…Unlike the model yeast Saccharomyces cerevisiae , targeted allele replacement in A. fumigatus is complicated by low rates of homologous recombination and the fact that replacement cassettes require long homology arms (LHA) of c. 1 kb ( Stafa et al 2017 ). Improvement in the frequency of homologous recombination can be achieved by employing strains deficient in the non-homologous end-joining (NHEJ) pathway (Δ ku80 , Δ ku70, and Δ lig4 strains) ( da Silva Ferreira et al, 2006 , Ishibashi et al, 2006 ).…”

Novel Approach in the Construction of Bioethanol-Producing Saccharomyces cerevisiae Hybrids

“…Therefore, frequent TCD in these strains could be a consequence of some mutations, change in gene expression and/or protein activity that remains to be identified. From this point of view, it should be noted that fidelity of gene targeting and spectrum of genetic events strongly depend on the transformation procedure used to deliver the transforming DNA to the yeast cell ( 30 ) as well as on the influence of e.g. EXO1 and SGS1 genes on both transformation efficiency and gene targeting fidelity ( 28 , 37 ).…”

“…Lithium acetate transformation was done as described previously ( 28 ) but since it was observed that the efficiency of transformation in natural isolates was lower than in commonly used laboratory strains, cells were first allowed to recover for 30 min in rich YPD medium and then plated on selective SC-Ura plates. Isolation of the genomic DNA was performed as described previously ( 29 ) and molecular analysis of transformants was done by Southern blotting ( 30 ) using dioxigenin (DIG)-labelled ADE2 gene (Roche, Darmstadt, Germany). Statistical analysis of the spectra of transformation events was done using two-tailed Pearson’s chi-squared test ( χ 2 ).…”

Toolbox for Genetic Transformation of Non-Conventional Saccharomycotina Yeasts: High Efficiency Transformation of Yeasts Belonging to the Schwanniomyces Genus

“…The transformation of all yeast species in this work was performed using electroporation protocol described in [ 3 ], which was originally developed for transformation of B. bruxellensis. Yeast genomic DNA was isolated as in [ 20 ], and Southern blot was performed as in [ 21 ]. The digoxigenin-labelled probe was synthesized with the DIG DNA Labelling Mix (Roche, Mannheim, Germany), using as a template the PvuII-EcoRI fragment of pRS500 plasmid ( Figure 1 ), thus labelling the gene bla and ori.…”