sequence reads of non fluorescent transformants of the ScEEB1 deletion in Saccharomyces pastorianus CBS1483

Saccharomyces pastorianus is not a classical taxon, it is an interspecific hybrid resulting from the cross of Saccharomyces cerevisiae and Saccharomyces eubayanus. Exhibiting heterosis for phenotypic traits as wort alpha-oligosacharides consumption and fermentation at low temperature, it has been domesticated to become the main work-horse of the brewing industry.Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR induced double strand break is unpredictable and preferentially uses the homoeologous chromosome as template preventing targeted introduction of the desired repair construct. Here, we demonstrate that lager hybrids can be edited with near 100% efficiency at carefully selected landing sites on the chimeric SeScCHRIII. The landing sites were systematically selected and evaluated for i) absence of loss of heterozygosity upon CRISPR-editing, ii) efficiency of the gRNA, and iii) absence of effect on strain physiology. Successful examples of highly efficient single and double genes integration illustrated that genome editing can be applied in interspecies hybrids paving the way to a new impulse to lager yeast strain development.Sequencing of four non fluorescent transformants of the ScEEB1 deletion transformation were randomly selected and subjected to whole genome sequencing (WGS).Sequencing analysis confirmed that transformants failed to incorporated the supplied repair fragment, but instead repaired the DSB with the homoeologous S. eubayanus CHRXVI as template for HDR resulting in loss of heterozygosity. Each transformant exhibited a different pattern: colony 1 , colony 4 and colony 3 were repaired by a 5 kbp, 10 kbp and 26-kbp region surrounding the ScEEB1 gene, while in colony 6 over 700 kbp from the ScCHRXVI was replaced by the homologous sequence from the SeCHRXVI sequence and only the subtelomeric regions of ScCHRXVI were retained .