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A team led by researchers at ��ɫtv Langone Health has built nine new synthetic yeast chromosomes, swapping out a key organism’s genetic material for engineered replacements.

The assembly of the chromosomes, and a set of related biological insights, are among the findings published online November 8 in 10 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 Saccharomyces cerevisiae. Yeast are simpler and easier to study than human cells, and they 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 eight 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.

Along with the eight newly reported synthetic chromosome assemblies related to versions that occur in nature, one new study () described the building of a nonnaturally occurring chromosome housing the genes for all 275 transfer ribonucleic acids (tRNAs) in Baker’s yeast. tRNAs are essential for the building of proteins, and the tRNA neo-chromosome promises to improve the stability of the synthetic yeast genome and to ensure the accurate construction of proteins built in such yeast, increasing their survival chances.

“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 , the Sol and Judith Bergstein Director of the at ��ɫtv Langone, whose team was responsible for 5 of the 10 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.

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, says Dr. Boeke, also a professor in the . 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, , 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, , described some completely new chromosome gymnastics, led by , a research assistant professor of biochemistry and molecular pharmacology and member of the Institute for Systems Genetics. 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.

Dr. Zhang’s team developed a new technology that uses 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 Dr. 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 postdoctoral 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. 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 during their successful build of synIX, and published as part of the new packet of papers ().

Successful transfer of the largest single synthetic chromosome, synIV, into a single yeast strain that already had 7.5 synthetic chromosomes in place by Dr. Zhao and team resulted in a strain that now carries more than 50 percent 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 synthetic 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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