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Tom Drake

Singer Instruments, Roadwater, Somerset, TA23 0RE, UK.

Introduction

Saccharomyces cerevisiae has long been utilised as a model organism due to the replicable nature of eukaryotic processes. Recent advances in genome editing, with tools such as CRISPR becoming more efficient and controlled, have enabled further advances into the manipulation of S. cerevisiae including modifications to the karyotype. Recent publications in Nature from Shao et al.¹ and Luo et al.² have done just this and opened the door to further research in chromosome biology and the evolutionary nature of chromosome development and function.

S. cerevisiae

Wild type S. cerevisiae contains 16 chromosomes, each with a distinct set of genes, a centromere and a telomere at each end. How this species came to have 16 chromosomes is a question not fully understood. For example, we know some of our closest ancestors in primates have 24 pairs of chromosomes, yet we only have 23 pairs. This is due to an ancestral fusion in what we now know as Chromosome 2³. The number of chromosomes that a species has is unlikely to be chance, and more likely to be a product of an evolutionary advantage, but what happens if a species had less chromosomes?

Two groups simultaneously investigated what would happen to S. cerevisiae if they reduced the number of chromosomes, without removing any essential genes.

The two groups; from Institute for Systems Genetics, NYU Langone Health, USA, and Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, China both published their results in Nature on August 1st 2018. Both groups simultaneously worked on reducing the number of chromosomes whilst maintaining the genome of S. cerevisiae. Utilising CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which is one of the most aggressive engineering strategies, Luo et al. removed telomeres and one centromere at a time to reduce the number of chromosomes from n=16 stepwise to n=2. This process produced a variety of strains with varying chromosomal numbers which are beneficial for further genotype and phenotype research.

Shao et al. created a single linear chromosome containing each of the 16 chromosomes, through successive end-to-end fusions and centromere removal. Interestingly, Luo et al. were unable to produce a stable yeast with only one chromosome, yet Shao et al. were. This is potentially due to the differing techniques used to edit the genome, or something as simple as the order of the fusions contributing to the success of the strain. Further research into this would prove interesting as this may suggest why one method was more successful.

Great, now we have n=1 and n=2, so what?

Now the strains had been engineered to only contain one, or two, chromosomes, the question was to investigate what difference this made compared to the wild type (n=16). As the role that chromosomes can play in the regulation of genes is not yet fully understood, they were careful to investigate gene expression in their new strains. Surprisingly, there was very little difference identified in the transcriptome or phenome of the n=1 strain and n=16. The other group, with n=2, reported modest transcriptomic changes although growth was shown with no major defects. This research suggests that a reduction in karyotype may have much less of an impact than first thought.

What if they mate?

S. cerevisiae grows as a haploid, meaning it only has one copy of each chromosome – humans are diploid, in that we have two copies of each chromosome. Haploid yeast can grow asexually, and continue to be clonal, or they can mate sexually and form diploid yeast before sporulating into haploid spores. This process transfers genomic material through genetic recombination. What happens if two strains with differentiating numbers of chromosomes mate sexually?

Luo et al. investigated this, and with varying results, showed that as the number of chromosomes drops below 16, the viability of spores decreases. When n=12, the viability of spores was less than 10%. When the wild type crossed with n=8, the viability of spores was less than 1%. Conversely, these strains could mate with others with the same number of chromosomes and produce viable tetrads and spores. This suggests that eight chromosome fusion events isolate these strains from reproducing and therefore enable isolated genetic evolution potentially leading to new species.

What have we learnt?

From the genetic engineering of S. cerevisiae chromosomes, reducing the total number from 16 to 1, we’ve seen that there are no major reductions in transcriptomes or phenomes. The reduction in chromosome number has also been investigated in terms of mating ability, with spore viability decreasing as the difference in chromosome number increases. Although, each strain could still mate with an identical chromosome number, and produce viable spores.

This research has created the basis for further investigation into the function, structure and purpose of chromosomes and the role that they play in a variety of essential processes in the cell. Alongside paving the way for the study of chromosome evolution and the significance of chromosome number within S. cerevisiae.

Jef Boeke and his laboratory at the Institute for Systems Genetics, NYU Langone Health, New York, USA, utilise an MSM tetrad dissection microscope to dissect spores, and have more recently added a SporePlay+ tetrad dissection microscope, a ROTOR HDA colony manipulation robot and a PhenoBooth colony counter to their laboratory.

References

1. Yangyang Shao, Ning Lu, Zhenfang Wu, Chen Cai, Shanshan Wang, Ling-Li Zhang, Fan Zhou, Shijun Xiao, Lin Liu, Xiaofei Zeng, Huajun Zheng, Chen Yang, Zhihu Zhao, Guoping Zhao, Jin-Qiu Zhou, Xiaoli Xue & Zhongjun Qin. Creating a functional single-chromosome yeast. Nature (2018)
2. Jingchuan Luo, Xiaoji Sun, Brendan P. Cormack & Jef D. Boeke. Karyotype engineering by chromosome fusion leads to reproductive isolation in yeast. Nature (2018).
3. IJdo JW, Baldini A, Ward DC, Reeders ST, Wells RA. Origin of human chromosome 2: an ancestral telomere-telomere fusion. Proc Natl Acad Sci U S A. (1991);88(20):9051-5.

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Amber Leckenby¹
¹ Singer Instruments, Roadwater, Somerset, TA23 0RE, UK

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INTRODUCTION

Systematic libraries have proven to be invaluable to genome-wide studies within yeast – examples include the yeast knockout collection and the yeast GFP library (find more libraries here). Each library has enabled novel insight into all aspects of yeast biology yet, their value is often overshadowed by the enormous effort required to make them. The large cost and lengthy laborious workflow of endless transformations, clone picking and validation steps often deter scientists from tackling other emerging biological questions.

Anton Khmelinskii and Matthias Meurer in Michael Knop’s lab at the University of Heidelberg developed a seamless gene tagging method^[1] to help alleviate the problems faced during library construction. Together with Ido Yofe and Uri Weill in Maya Schuldiner’s lab at the Weizmann Institute of Science, Israel, they have further developed this method into the SWAp-Tag method. By utilising the ROTOR, this method allows rapid library construction in just three weeks ^[2].

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Prof. Maya Schuldiner Weizmann Institute of Science, Israel

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SWAp-Tag (SWAT) TECHNOLOGY

The SWAp-Tag (SWAT) method relies on the generation of an initial acceptor library. This acceptor library acts as a template that can be swapped into other libraries of choice in a “plug and play” manner (Figure. 1).

The library construction is unique in the fact that it requires a one-off traditional construction of an acceptor strain library with an acceptor module inserted at a known genomic location. This acceptor module can be replaced by a new tag, promoter or other desired genomic sequence by mating of the acceptor library with a donor strain expressing the desired module. The result is an unlimited number of new libraries created easily, accurately and cost-effectively.

Figure 1. The SWAT strategy enables rapid and straight-forward generation of systematic libraries. (a) Integration of the SWAT acceptor module to tag proteins at the N’ terminus. (b) Utilisation of the ROTOR to mate the acceptor strains with donor strain to create library of choice. Further utilisation of the ROTOR to select for haploid spores and induce I-SceI expression. (c) Examples of potential donor plasmids (top) that could be used to create gene tagged libraries (bottom).

SWAT TECHNOLOGY IS FAST, FLEXIBLE AND FREELY AVAILABLE

The Schuldiner and Knop labs have created an innovative and extremely rapid method for library construction by generating a library that has the ability to incorporate different modules easily, instead of creating a new library from scratch for each module. After acquisition or construction of an acceptor library, a new library can be generated in just three weeks and can be immediately used in a wide range of genome wide studies. The Schuldiner lab have already created an original acceptor N’-SWAT library which is N-terminally tagged with constitutively expressed GFP. In addition, two more libraries have been made from the original acceptor library using the SWAT method: an N’ mCherry-tag library and an N’ seamless GFP library. The published SWAT library is now freely available in GFP-tag, mCherry-tag or seamless GFP flavour from Prof. Maya Schuldiner.

The bottlenecks in this method are the mating and selection steps. The Schuldiner and Knop labs were able to use the Singer Instruments ROTOR to alleviate these bottlenecks. As such, the ROTOR was used for all handling of strains, mating and sporulating procedures, tag-swap selections/counter-selection and in library screens for the selection of successful module integration.

“Creation of new libraries would not have been possible without our Singer ROTOR robot”.
Prof. Maya Schuldiner – 2017

This swift process means that scientists can afford to be flexible in the libraries that they create and visualise never before seen proteins. Tag-mediated localisation problems have been solved by the SWAp-TAG method as it enables ORFs to be tagged at either the 5’ or 3’ end to minimise any mis-localisation or protein-destabilisation effects.

WHAT’S NEXT FOR THE SWAT STRATEGY: APPLICATIONS AND IMPLICATIONS?

Theoretically, any tag can be used to construct a new library from this N’-SWAT acceptor library. This tag could be different coloured fluorophores for co-localization studies or complementation tags for measuring protein-protein interactions. Once a C’-SWAT acceptor library has been generated, any section of the 5’- or 3’-end of a gene can be modified whether it be a promoter, UTR or other non-coding DNA to quantify transcription and translation effects. Half-lives can be studied using timer fluorophores or a pull down tag can be attached to isolate proteins.

“The sky is this limit and now only each labs’ imagination is the problem”.
Prof. Maya Schuldiner – 2017

The SWAT method for library generation frees up valuable time, allowing researchers to design impactful experiments. The Schuldiner lab are already using the N’ SWAT library in co-localization screens, overexpression screens and for looking at interactomes in vivo. As Professor Schuldiner has said herself, “The fun never ends!”.

Discover how the ROTOR can help you too!

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Synthetic Yeast Chromosome Manipulation