Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes

doi:10.1016/j.jbiosc.2012.01.013

Journal of Bioscience and Bioengineering

Volume 113, Issue 6, June 2012, Pages 675-682

https://doi.org/10.1016/j.jbiosc.2012.01.013 Get rights and content

Abstract

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18 kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.

Section snippets

Strains, media, and transformation

S. cerevisiae strain FY834 (17) (MATα his3Δ200 ura3-52 leu2Δ1 lys2Δ202 trp1Δ63) was the principal strain used in the study; other strains that were used are listed in Table 1. Yeast cells were grown on YPDA medium containing 1% yeast extract, 2% peptone, 2% glucose, and 0.04% adenine (Sigma-Aldrich, St. Louis, MO, USA) or SD medium containing 2% glucose and 0.67% Difco™ yeast nitrogen base without amino acids (YNB; BD Biosciences, Sparks, MD, USA). SG medium was also used in this study and

Splitting chromosome IV into 11 new chromosomes

We derived the yeast stock ZN1 (Fig. 1A) in our laboratory from FY834, and after splitting chromosome IV into 2 derivative chromosomes at the LYS4 and LYS14 loci (12; data not shown), we subsequently derived strain ZN3. Using ZN3 as the starting strain, we sought to further split the derivatives of chromosome IV into 11 mini-chromosomes with the PCS method (8). For this purpose, the target sequence was extended to 650 bp using overlap-extension PCR as follows: a 30 base overlap sequence was

Acknowledgments

This work was supported by Grant-in-Aid for Scientific Research (B) 12460044 (to S. H.), (B) 15380064 (to S. H.), Grant-in-Aid for challenging Exploratory Research 19658132 (to S. H.), and Grant-in-Aid for Young Scientists (B) 23780080 (to M. S.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. This work was also carried out as a part of the project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of METI and NEDO.

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Single-chromosome fission yeast models reveal the configuration robustness of a functional genome
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Citation Excerpt :
The single-chromosome and two-chromosome budding yeast Saccharomyces cerevisiae strains artificially constructed displayed drastic changes in global chromosome structures, but show little change in gene expression profiles and exhibit no apparent defects in various phenotypes (Luo et al., 2018; Shao et al., 2018). Conversely, increasing chromosome numbers from 16 to 21 or 30 by splitting native chromosomes in Saccharomyces cerevisiae also seems phenotypically inert (Ueda et al., 2012; Widianto et al., 2003). Whether strong tolerance to chromosome configuration changes is a ubiquitous attribute of eukaryotic genomes is unknown.
In eukaryotic organisms, genetic information is usually carried on multiple chromosomes. Whether and how the number and configuration of chromosomes affect organismal fitness and speciation remain unclear. Here, we have successfully established several single-chromosome fission yeast Schizosaccharomyces pombe strains, in which the three natural chromosomes have been fused into one giant chromosome in different orders. Chromosome fusions accompanied by the deletions of telomeres and centromeres result in the loss of chromosomal interactions and a drastic change of global chromosome organization, but alter gene expression marginally. The single-chromosome strains display little defects in cell morphology, mitosis, genotoxin sensitivity, and meiosis. Crosses between a wild-type strain and a single-chromosome strain or between two single-chromosome strains with different fusion orders suffer defective meiosis and poor spore viability. We conclude that eukaryotic genomes are robust against dramatic chromosomal reconfiguration, and stochastic changes in chromosome number and genome organization during evolution underlie reproductive isolation and speciation.
Design, building, and challenges in synthetic genomics
2022, New Frontiers and Applications of Synthetic Biology
To a large extent, synthetic biology became possible due to the advances in our understanding of genomes and the evolution of technologies that have enabled the assembly of large DNA molecules and the creation of artificial genomes. These technologies have ushered in a new era of synthetic genomics. The application of these technical advances combined with an understanding of genes and genome structure has made it possible to create artificial genomes. Artificial genomes are based on the overarching principle of creating programmable self-sustainable genomes that confer novel biological properties to the recipient genomes and cells. The process of minimization, design, and building of artificial genomes is complex and iterative. It requires substantial tweaking of genes and chromosomes, regulatory elements, and machinery and has led to rich insights into the design principles of genome organization that will be discussed in this chapter.
CRISPR-PCD and CRISPR-PCRep: Two novel technologies for simultaneous multiple segmental chromosomal deletion/replacement in Saccharomyces cerevisiae
2020, Journal of Bioscience and Bioengineering
Genome manipulation, especially the deletion or replacement of chromosomal regions, is a salient tool for the analysis of genome function. Because of low homologous recombination activity, however, current methods are limited to manipulating only one chromosomal region in a single transformation, making the simultaneous deletion or replacement of multiple chromosomal regions difficult, laborious, and time-consuming. Here, we have developed two highly efficient and versatile genome engineering technologies, named clustered regularly interspaced short palindromic repeats (CRISPR)-PCR-mediated chromosomal deletion (PCD) (CRISPR-PCD) and PCR-mediated chromosomal replacement (CRISPR-PCRep), that integrate the CRISPR-associated protein 9 (Cas9) genome editing system (CRISPR/Cas9) into, respectively, the PCD method for chromosomal deletion and our newly developed PCRep method for chromosomal replacement. Integration of CRISPR induces double strand breaks to activate homologous recombination, and thus enhances the efficiency of deletion by PCD and replacement by PCRep, enabling multiple chromosomal regions to be manipulated simultaneously for the first time. Our data show that CRISPR-PCD can delete two internal or terminal chromosomal regions, while CRISPR-PCRep can replace triple chromosomal regions simultaneously in a single transformation. Colony PCR analysis of structural alterations showed that triple replacement of four different sets of chromosomal regions was successful in 83%–100% of transformants analyzed. These novel genome engineering technologies, which greatly reduce time and labor for genome manipulation, will provide powerful tools to facilitate the simultaneous multiple deletion and replacement of chromosomal regions, enabling the rapid analysis of genome function and breeding of useful industrial yeast strains.
Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae
2016, Analytical Biochemistry
Citation Excerpt :
After several repeated chromosome operations, a high number of foreign sequences that remain may result in chromosome instability. The PCR-mediated approach [3] combines two-step PCR and one transformation per splitting event with the Cre/loxP system for marker rescue, and using this method, chromosomes have been successfully split collectively into minichromosomes that were occasionally lost during mitotic growth in various combinations. This innovative method is simple and efficient, but the minichromosomes have uncertainty.
Large chromosomal modifications have been performed in natural and laboratory evolution studies and hold tremendous potential for use in foundational research, medicine, and biotechnology applications. Recently, the type II bacterial Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR/Cas9) system has emerged as a powerful tool for genome editing in various organisms. In this study, we applied the CRISPR/Cas9 system to preform large fragment deletions in Saccharomyces cerevisiae and compared the performance activity to that of a traditional method that uses the Latour system. Here we report in S. Cerevisiae the CRIPR/Cas9 system has been used to delete fragments exceeding 30 kb. The use of the CRISPR/Cas9 system for generating chromosomal segment excision showed some potential advantages over the Latour system. All the results indicated that CRISPR/Cas9 system was a rapid, efficient, low-cost, and versatile method for genome editing and that it can be applied in further studies in the fields of biology, agriculture, and medicine.
Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering inSaccharomyces cerevisiae
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Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected—a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.
Spindle architecture constrains karyotype in budding yeast
2023, bioRxiv

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^†: The first two authors contributed equally to this work.

^§: Present address: Central Laboratories for Frontier Technology, KIRIN Holdings Co., Ltd., Yokohama, Kanagawa, Japan.

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Journal of Bioscience and Bioengineering

Abstract

Section snippets

Strains, media, and transformation

Splitting chromosome IV into 11 new chromosomes

Acknowledgments

References (23)

A yeast artificial chromosome-splitting vector designed for precise manipulation of specific plant chromosome region

J. Biosci. Bioeng.

Repeated chromosome splitting targeted to delta sequences in Saccharomyces cerevisiae

J. Biosci. Bioeng.

Creating a Saccharomyces cerevisiae haploid strain having 21 chromosomes

J. Biosci. Bioeng.

Construction and characterization of single-gene chromosomes in Saccharomyces cerevisiae

J. Biosci. Bioeng.

A versatile and general splitting technology for generating targeted YAC subclones

Appl. Microbiol. Biotechnol.

Chromosome XII context is important for rDNA function in yeast

Nucleic Acids Res.

A polymerase chain reaction-mediated yeast artificial chromosome-splitting technology for generating targeted yeast artificial chromosomes subclones

Methods Mol. Biol.

Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA

Biotechniques

Large scale deletions in the Saccharomyces cerevisiae genome create strains with altered regulation of carbon metabolism

Appl. Microbiol. Biotechnol.

PCR-mediated repeated chromosome splitting in Saccharomyces cerevisiae

Biotechniques

Chromosome-shuffling technique for selected chromosomal segments in Saccharomyces cerevisiae

Appl. Microbiol. Biotechnol.

Cited by (18)

Single-chromosome fission yeast models reveal the configuration robustness of a functional genome

Design, building, and challenges in synthetic genomics

CRISPR-PCD and CRISPR-PCRep: Two novel technologies for simultaneous multiple segmental chromosomal deletion/replacement in Saccharomyces cerevisiae

Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae

Stabilization of mini-chromosome segregation during mitotic growth by overexpression of YCR041W and its application to chromosome engineering inSaccharomyces cerevisiae

Spindle architecture constrains karyotype in budding yeast