Researchers complete 8 additional synthetic yeast chromosomes, plus a bonus tRNA neochromosome!

An international team of researchers led by Prof. Jef Boeke at NYU Langone Health has built eight new synthetic yeast chromosomes, swapping out a key organism’s genetic material for engineered replacements, and an bonus tRNA neochromosome. Synthetic chromosomes were constructed by groups spanning the globe (alphabetical order by country and organization, the sun never sets on Sc2.0!):

• Australia: Australian Wine Research Institute, Macquarie University
• China: BGI, Shenzhen Institute of Advanced Technology (SIAT), Tianjin University, Tsinghua University
• Japan: Tokyo Institute of Technology (TITech)
• Singapore: National University of Singapore (NUS)
• United Kingdom: Imperial College, University of Edinburgh, University of Manchester
• United States: GenScript, Johns Hopkins University (JHU), New York University (NYU)

For an overview of the global collaboration, see this commentary in Cell Genomics (2023).

The assembly of the chromosomes, and a set of related biological insights, are among the findings published online November 8 in ten papers in the journals Cell, Molecular Cell, and Cell Genomics.

The new studies are the latest from the Synthetic Yeast Project (Sc2.0), the consortium that, after 15 years of work by more than 250 researchers worldwide, is getting close to its goal: build synthetic versions of all 16 chromosomes—the structures that contain DNA—for the one-celled microorganism Baker’s yeast, known as S. cerevisiae. Yeast are simpler and easier to study than human cells, and similarly package their genetic material in linear superstructures called chromosomes inside cell nuclei.

Although bacterial and viral chromosomes have been synthesized previously, this would be the first synthetic genome in a eukaryotic cell, a class that includes human cells, has several chromosomes per cell, and has structural complexities not present in bacteria or viruses.

In 2011, researchers built the first synthetic chromosome arm, synIXR, which launched the international project. In 2014, Sc2.0 reported the building of the first synthetic yeast chromosome (synthetic chromosome 3, or synIII), and five additional chromosomes (synII, synV, synVI, synX and synXII) followed by 2017. Along with the 8 reported today, the team has also already assembled the remaining two chromosomes of the 16, with those last results expected to publish before the end of 2023.

“Assembling all yeast chromosomes from the ground up is a monumental feat, with Sc2.0 scientists having made thousands of design changes to the yeast’s genetic instructions in a new era of high-speed experimentation,” said Jef Boeke, PhD, director of NYU Langone’s Institute for Systems Genetics, whose team was responsible for five of the ten publications released today. “Our maturing research platform – a customized, fully functioning synthetic lifeform – promises to contribute to a near-future wave of solutions to previously intractable challenges in medicine, the environment, and bioenergy.”

Specifically, such advances may include making yeast that have been engineered to produce antibiotics or vaccines able to do so in much greater amounts, which would be a boon to biomedicine. Other projects promise to improve the efficiency of biofuel production, yield more effective yeast-based tools that remove pollutants from soil and water, or fight starvation by supplementing livestock feed.

International collaboration creates a tRNA neochromosome

Researchers in the Manchester Institute of Biotechnology (MIB) at The University of Manchester have created the tRNA neochromosome – a chromosome that is new to nature. It forms part of a wider project (Sc2.0).

The tRNA neochromosome and an additional synthetic chromosome are a culmination of 10 years of research from an international consortium of scientists led by Professor Patrick Cai and The University of Manchester, and mark a new chapter in engineering biology. The University of Manchester’s research also features on the front covers of Cell and Cell Genomics.

Prof Cai, Chair in Synthetic Genomics at The University of Manchester and international coordinator of Sc2.0 project, said: “This is an exciting milestone when it comes to engineering biology. While we have been able to edit genes for some time, we have never before been able to write a eukaryote genome from scratch. This work is fundamental to our understanding of the building blocks of life and has the potential to revolutionise synthetic biology which is fitting as Manchester is the home of the Industrial Revolution. Now, we’re at the forefront of the biotechnological revolution too. What’s remarkable about this project is the sheer scale of collaboration and the interdisciplinarity involved in bringing it to fruition. We’ve brought together not only our experts here in the MIB, but also experts from across the world in fields ranging from biology and genomics to computer science and bioengineering."

Dr Daniel Schindler, one of the two lead authors of Manchester’s contribution and now group leader at the Max Planck Institute for Terrestrial Microbiology and the Center for Synthetic Microbiology (SYNMIKRO) in Marburg, said: “The international Sc2.0 is a fascinating, highly interdisciplinary project. It combines basic research to Commented [PC1]: Note - we actually got both covers!expand our understanding of genome fundamentals, but also paves the way for future applications in biotechnology and drives technology developments. The international and inclusive nature of the project has unleashed the science and seeded future collaborations and friendships. The Manchester Institute of Biotechnology, with its excellent research environment and open space, has always facilitated this.”

The tRNA neochromosome is used to house and organise all 275 nuclear tRNA genes from the yeast and will eventually be added to the fully synthetic yeast where the tRNA genes have been removed from the other synthesised chromosomes. Unlike the other synthetic chromosomes of the Sc2.0 project, the tRNA neochromosome has no native counterpart in the yeast genome. It was designed using AI assisted, computer-assisted design (CAD), manufactured with state-of-the- art roboticized foundries, and completed by comprehensive genome-wide metrology to ensure the high fitness of the synthetic cells. Next, the researchers will work together to bring all the individual synthetic chromosomes together into a fully synthetic genome. The final Sc2.0 strain will not only be the world’s first synthetic eukaryote, but also the first one to be built by the international community.

“The potential benefits of this research are universal – the limiting factor isn’t the technology, it’s our imagination”, says Prof Cai.

Insights Along the Way

Exactly how chromosomes are packaged inside cells is known to impact the flow of genetic information that shapes each cell’s behavior, Boeke says. Incorrect folding of chromosomes can disrupt 3D interactions between the long stretches of DNA packaged in chromosomes, causing the transmission of faulty instructions linked to many diseases. However, results from Sc2.0 investigators today demonstrate that global changes in the 3D structure of the genome can also be surprisingly well tolerated.

Published today in Cell Genomics, another of the new papers, Luo et al, provided evidence of the considerable tolerance of the yeast genome to 3D structural changes. As the team built the first-ever synthetic fusion chromosome, synI-synIII, a combination of two natural chromosomes, the yeast containing it continued to grow despite their significantly altered 3D structure.

Another new paper, published in Molecular Cell, described some completely new chromosome gymnastics, led by Weimin Zhang, PhD, research assistant professor in the Institute for Systems Genetics at NYU Langone Health. After building synIV, the largest Sc2.0 chromosome, investigators engineered the first synthetic chromosome with an altered 3D conformation by flipping the ‘arms’ of the chromosome inside out. Surprisingly, this drastic structural change in the core of the nucleus also caused minimal changes in the activity of genes.

Zhang’s team developed a new technology that use designer proteins to tether synIV to the membrane that separates the nucleus from the rest of the cell, a much more drastic spatial change never before attempted. In the resulting alteration of 3D structure, expression of most of the synIV genes was silenced, without changing a single letter of DNA code. These studies are helping investigators tease out the types of structural changes that have deleterious gene expression and growth versus those that do not. “Manipulating synIV’s 3D structure paves the way for fully predictable 3D genome engineering—a major advance in synthetic biology enabling us to define how synthetic genomes are packaged and regulated within a cell,” noted Zhang.

Finally, the Luo et al. team uncovered new mechanisms critical for the transmission of small chromosomes during meiosis, a specialized type of cell division that occurs in sexually reproducing organisms that in humans results in egg and sperm cells. Understanding these processes provides clues for understanding how chromosomes fail to separate in conditions such as Down syndrome.

“The coordinated work of the many Sc2.0 investigators and trainees globally has reached the end of the beginning—the complete synthesis of a synthetic yeast genome,” said lead author, Yu Zhao, PhD, a post-doctoral fellow in the Institute for Systems Genetics. “By using our new methods, we are learning new twists on the rules of life.”

The team’s challenge now, after having assembled all 16 synthetic yeast chromosomes, is to consolidate them into a single living yeast strain, the authors said. Zhao and colleagues just published in Cell their significant progress on this front by incorporating 6.5 synthetic yeast chromosomes into one yeast cell using an established, but tedious method. Realizing they needed a more scalable approach, the team used a technique called chromosome substitution, developed by Sc2.0 colleagues in the Boeke Lab during their successful build of synIX, and published as part of the new packet of papers (McCulloch et al., Cell Genomics).

Successful transfer of the largest single synthetic chromosome, synIV, into a single yeast strain that already had 7.5 synthetic chromosomes in place by Zhao and team resulted in a strain that now carries more than 50% of the genetic information of the cells in synthetic form. Other advancements include the team’s development of a method, “CRISPR D-BUGS”, to identify “mistakes” in synthentic DNA sequences that impact the yeast’s ability to grow; and the use of SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) to rapidly create thousands of variant strains that can then be tested to see if they randomly developed useful properties.

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