Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast

Yue Shen, Feng Gao, Yun Wang, Ju Zheng, Jianhui Gong, Jintao Zhang, Zhouqing Luo, Daniel Schindler, Yang Deng, Weichao Ding, Tao Lin, Teem Swidah, Hongcui Zhao, Shuangying Jiang, Cheng Zeng, Shihong Chen, Tai Chen, Yong Wang, Yisha Luo, Leslie Mitchell, Joel S Bader, Guojie Zhang, Xia Shen, Jian Wang, Xian Fu, Junbiao Dai, Jef D Boeke, Huanming Yang, Xun Xu, Yizhi Cai

Preprint posted on 2 September 2022

Disomic yeast cells find relief from the stress of too much DNA by scrambling up the additional chromosome

Selected by Grace Heredge Thomas


Aneuploidy, an abnormality in chromosomal copy number, is typically the result of aberrant chromosome segregation during cell division. The resulting genomic instability can result in cell death, or in some organisms, can provide an opportunity for the cell to adapt to environmental conditions 1. In humans, aneuploidy is bad news; often the cause of embryo inviability, genetic disease, and tumorigenesis 2.

The effect of aneuploidy varies widely depending on the cell type and karyotype. This is challenging to study in multicellular model organisms, due to the difficulty in generating stable aneuploid cell populations. With decades worth of sophisticated molecular tools available, the budding yeast Saccharomyces cerevisiae is a widely used model for looking at the molecular mechanisms behind aneuploidy. By forcing mis-segregation, previous studies have been able to produce targeted aneuploid cells, and study the effect of various karyotypic changes 3. This preprint takes this a step further by investigating contributions of specific regions of chromosome VII to the fitness of aneuploid cells.

The Synthetic yeast genome project, or Sc2.0, led by Jef Boeke, is the first ever attempt to design and build a synthetic eukaryotic genome. The designer chromosomes have been streamlined and modified, with unstable repetitive motifs removed, and additional elements – like loxP sites and PCRTags – added in. These loxP sites are part of the SCRaMbLE system (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution), which facilitates the generation of combinatorial genomic diversity 4. In the presence of inducible Cre recombinase, DNA sequences flanked by loxP sites recombine with other loxP sites across the chromosome, resulting in inversions, deletions or insertions. By introducing loxP sites across the synthetic chromosomes, massive random chromosomal rearrangements can be induced at will. Using this system, the authors present a compelling new method to systematically investigate the effect of the loss or retention of sub-chromosomal regions on aneuploidy phenotypes.


Key findings

Synthetic chromosome VII can be stably maintained in a disomic yeast strain (n+1) and its presence leads to aneuploidy-specific phenotypes.

Using the standard design principles for producing a synthetic chromosome with SCRaMbLE capabilities, the authors constructed and debugged synthetic chromosome VII (synVII). At each stage of construction of the chromosome, cells containing the construct were assayed for fitness defects in a process called debugging. These growth defects could be caused by modifications to gene flanking sequences. To restore fitness that section of synVII had to be redesigned.

In order to generate an aneuploid strain with synVII, the authors first made an n+chrVII disomic strain. This chrVII-specific aneuploid yeast strain (YSy140 n+chrVII) was generated by inserting a galactose inducible promoter adjacent to chrVII CEN7, resulting in non-disjunction of chrVII. YSy140 and the strain containing synVII (YSy105) were mated and sporulated to finally obtain a disomic strain with one copy of native chrVII and one copy of synVII (YSy142 n+synVII). This strain was verified with whole genome sequencing and exhibited aneuploidy-specific phenotypes, namely increased sensitivity to hydroxyurea (an inhibitor of ribonucleotide reductase), cycloheximide (a protein synthesis inhibitor), and methyl methanesulfonate (a DNA-damaging agent).

969 recombination events were detected in 219 SCRaMbLEd aneuploid yeast strains

To induce SCRaMbLE in synVII, a Cre recombinase expression plasmid was transformed into YSy142. Induction of Cre expression resulted in recombination across the genome between segments flanked by loxP sites. SCRaMbLEd aneuploid derivatives were recovered on selective agar medium and analyzed by genome sequencing.

Overall, 989 recombination events were identified in 291 strains; 62% were deletions, 29% were inversions and 9% were duplications. On average, there were 42 events per strain. The sequence adjacent to the centromere was the most consistently retained sequence likely due to the presence of the selection marker LEU2 by the centromere. The authors speculated that it is possible that genes near the telomeres could be toxic when multiple copies are present, as these regions were frequently absent from the SCRaMbLEd strains.

Fig 1- The SCRaMbLE strategy produced strains with rearranged synVII that could be rescued on agar selection media. 291 SCRaMbLEd synVII were sequenced and the sequence was reconstructed. The genomes of these strains had unique deletions, inversions or duplication events.


SCRaMbLEing of the SynVII chromosome rescued aneuploidy phenotypes

Relative fitness of the SCRaMbLEd strains was calculated by comparing average colony size of >200 colonies to the original YSy142 aneuploid colonies. The SCRaMbLEd colonies showed improved fitness in the presence of cycloheximide, varying from 6.6%-80%. The authors identified two types of structural conformation of the SCRaMbLEd synVII (circular and linear). 89% of the 219 strains had the circular form of synVII. Some of these strains had lost most of the chromosomal arms, retaining just 1-19% of synVII. These strains had a higher fitness recovery rate (40%-60%) (figure 2). On the other hand, the authors found that 18/24 of strains that had retained <50% of the synVII had a more modest recovery rate (average of 32.4%). This supports the “mass action of genes hypothesis”, suggesting that aneuploidy phenotypes are the result of copy alterations of many genes that may not individually have observable phenotypes.

Fig 2- General improvement of fitness of the top 18 SCRaMbLEd strains in 5 representative conditions compared to parental disomic strain. Each dot represents one strain’s recovery rate calculated based off the size of >200 single colonies. From left to right: YPD, YDP + hydroyurea (HU 100mM), YPD + cycloheximide (Cyc 0.01µg/mL), YPD + DL-Dithiothreitol (DDT 2.5mM), YPD + methyl methane sulfone (MMS 0.01%).


Removal of a specific 20Kb region led to improved fitness and was linked to up-regulation of translation.

Using a chromosome-wide association analysis, the authors identified a 20kb deletion present in several strains that may play a role in fitness recovery in aneuploidy strains. In addition, a proteomic analysis of 5 strains showed that protein synthesis and ribosome biogenesis were up-regulated. The authors suggested that this result implies that there are specific genes that can cause aneuploidy phenotypes when duplicated. Their findings therefore provide evidence for both the “mass action of genes” theory and the idea that there are key genes that are toxic when present in multiple copies 5.


Why I chose this study:

Synthetic biology provides creative approaches to explore molecular mechanisms. I like this study because it attempts to unpick an incredibly complicated phenotype. By producing hundreds of scrambled-up, aneuploid strains the authors have attempted to reach the level of diversity seen in aneuploidy in nature. I appreciate the systematic approach that allows us to focus solely on one chromosome at a time to identify particular genes and regions, and I look forward to seeing the authors apply their SCRaMbLE approach to the other 15 chromosomes in yeast.


I find it curious that practically any rearrangement seems to improve the aneuploidy phenotypes, even when much of the chromosome sequence is retained. I would assume that the rearrangements cause a general disruption to the expression and function of the genes on synVII. It would be very interesting to further investigate the mechanisms behind some of these chromosomal rearrangements, and to see if any equivalent perturbations could be identified in aneuploid mammalian cells.


Questions for the author:

  • You show that aneuploidy phenotypes can be somewhat rescued by introducing the 20 kb deletion – identified in SCRaMbLEd strains – into synVII and wild type chrVII. Would you also expect to see an associated upregulation of protein synthesis and ribosome biogenesis? Perhaps additional proteomic analysis of these modified diploid strains could confirm the proposed causal relationship between the deletion and the upregulation of these processes.


  • In the case of the SCRaMbLEd chromosomes that retained most, or all the chromosome content, what mechanism do you propose causes the mitigation of aneuploidy phenotypes? Is it possible that SCRaMbLE causes loss of function of genes across the chromosomes?



  1. Kaya, A., Mariotti, M., Tyshkovskiy, A. et al. Molecular signatures of aneuploidy-driven adaptive evolution. Nat Commun 11, 588 (2020).


  1. Ben-David, U., Amon, A. Context is everything: aneuploidy in cancer. Nat Rev Genet 21, 44–62 (2020).


  1. Beach R R., Ricci-Tam C., Brennan CM., Moomau CA., Hsu P., Hua B., Silberman RE., Springer M., Amon A., Aneuploidy Causes Non-genetic Individuality., Cell 169, (2), (2017)


  1. Jia, B., Wu, Y., Li, BZ. et al. Precise control of SCRaMbLE in synthetic haploid and diploid yeast. Nat Commun 9, 1933 (2018).


  1. Bonney ME, Moriya H, Amon A., Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes.,Genes & Dev, 29, (2015)


Posted on: 17 October 2022


Read preprint (No Ratings Yet)

Have your say

Your email address will not be published.

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the cell biology category:


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage

Also in the genetics category:

Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University

This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.


List by Nándor Lipták

20th “Genetics Workshops in Hungary”, Szeged (25th, September)

In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link:


List by Nándor Lipták

2nd Conference of the Visegrád Group Society for Developmental Biology

Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)


List by Nándor Lipták

EMBL Conference: From functional genomics to systems biology

Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020


List by Jesus Victorino

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Zebrafish immunology

A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.


List by Shikha Nayar