Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts

被引:4
作者
Bennis, Nicole X. [1 ]
Kostanjsek, Matic [1 ]
van den Broek, Marcel [1 ]
Daran, Jean-Marc G. [1 ]
机构
[1] Delft Univ Technol, Dept Biotechnol, van der Maasweg 9, NL-2627 HZ Delft, Netherlands
关键词
Saccharomyces pastorianus; CRISPR-Cas9; Loss of heterozygosity; Landing sites; Genome modifications; Allo-aneuploid; ALPHA-ACETOLACTATE DECARBOXYLASE; LAGER BREWING YEAST; COPY NUMBER VARIATION; SACCHAROMYCES-CEREVISIAE; BREWERS-YEAST; ANTIVIRAL DEFENSE; GENE; SEQUENCE; DNA; PASTORIANUS;
D O I
10.1016/j.nbt.2023.04.001
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 such as wort alpha-oligosaccharide consumption and fermentation at low temperature, it has been domesticated to become the main workhorse of the brewing industry. Although CRISPR-Cas9 has been shown to be functional in S. pastorianus, repair of CRISPR-induced double strand breaks 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 gene integration illustrated that genome editing can be applied in interspecies hybrids, paving the way to a new impulse to lager yeast strain development.
引用
收藏
页码:49 / 62
页数:14
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