Abstract
In our previous study, a novel genome engineering technology, PCR-mediated chromosome duplication (PCDup), was developed in Saccharomyces cerevisiae that enabled the duplication of any desired chromosomal region, resulting in a segmental aneuploid. From one round of transformation, PCDup can duplicate a single chromosomal region efficiently. However, simultaneous duplication of multiple chromosomal regions is not possible using PCDup technology, which is a serious drawback. Sequential duplication is possible, but this approach requires significantly more time and effort. Because PCDup depends upon homologous recombination, we reasoned that it might be possible to simultaneously create duplications of multiple chromosomal regions if we could increase the frequency of these events. Double-strand breaks have been shown to increase the frequency of homologous recombination around the break point. Thus, we aimed to integrate the genome editing tool CRISPR/Cas9 system, which induces double-strand breaks, with our conventional PCDup. The new method, which we named CRISPR-PCDup increased the efficiency of a single duplication by up to 30 fold. CRISPR-PCDup enabled the simultaneous duplication of long chromosomal segments (160 kb and 200 kb regions). Moreover, we were also able to increase the length of the duplicated chromosome by up to at least 400 kb, whereas conventional PCDup can duplicate up to a maximum of 300 kb. Given the enhanced efficiency of chromosomal segmental duplication and the saving in both labor and time, we propose that CRISPR-PCDup will be an invaluable technology for generating novel yeast strains with desirable traits for specific industrial applications and for investigating genome function in segmental aneuploid.
Highlights
Saccharomyces cerevisiae is a model organism of immense industrial interest
We developed CRISPR-PCR-mediated chromosome duplication (PCDup) technology, which is an integration of PCDup with CRISPR/Cas9 that facilitates simultaneous and multiple duplication of chromosome segments in S. cerevisiae
We reasoned that integration of the CRISPR/Cas9 system with PCDup might increase the frequency of homologous recombination, thereby enabling simultaneous duplication
Summary
Saccharomyces cerevisiae is a model organism of immense industrial interest. It is known that many of the characteristics essential for the industrial application of S. cerevisiae, such as stress tolerance, are controlled by more than one gene (Swinnen et al 2012). Genome engineering technologies are required for the rapid and Hassan et al AMB Expr (2020) 10:27 in S. cerevisiae. One such method, named PCR-mediated chromosome duplication (PCDup), enables the duplication of any desired chromosomal region as an independent chromosome (Natesuntorn et al 2015). PCDup is able to duplicate chromosomal regions with lengths from 50 kb to 300 kb. Using PCDup, we discovered that segmental duplication of some chromosome regions leads to an enhanced resistant phenotype when the cells are grown under stress conditions. One round of duplication takes at least 11 days including confirmatory analysis and if the results is in failure, constructing stains by successive multiple chromosome duplications is both time consuming and laborious
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