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Synthetic Yeast Chromosome Progress Presented in New Publications

NEW YORK – Members of a large international team known as the Synthetic Yeast Project (Sc2.0) have generated synthetic versions of most chromosomes found in the yeast model organism Saccharomyces cerevisiae, along with a yeast strain that contains several of the synthetic chromosomes.

The Sc2.0 consortium, which includes hundreds of researchers from around the world, described details of its work in a series of papers published in the journals Cell, Cell Genomics, and Molecular Cell on Wednesday.

Building on the results from prior Sc2.0 research describing synthetic chromosomes and chromosome arms, the latest efforts led to eight new synthetic chromosomes, leaving just two synthetic versions of the S. cerevisiae chromosomes yet to be revealed.

"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," Yu Zhao, a postdoctoral researcher with the NYU Langone Health Institute for Systems Genetics, said in a statement.

In a paper published in Cell on Wednesday, first author Zhao and colleagues from NYU Langone Health, the European Molecular Biology Laboratory, and elsewhere used an advanced endoreduplication intercrossing strategy to mix and match yeast strains that each contained a different synthetic chromosome. The resulting yeast strain contained half a dozen complete synthetic chromosomes as well as a single synthetic chromosome arm.

"We decided that it was important to produce something that was very heavily modified from nature's design," senior and corresponding author Jef Boeke, a synthetic biologist with NYU Langone Health, said in a statement. "Our overarching aim was to build a yeast that can teach us new biology."

Along with long-read nanopore RNA sequencing and chromosome conformation capture (Hi-C) analyses of transcript isoform and 3D genome organization features, the team turned to a CRISPR-based gene editing approach to modify the sequences and search for so-called "bugs" stemming from the modifications.

"Thousands of genome-wide edits were introduced, including deletion of mobile elements and introns, relocation of [transfer RNAs], and swapping of stop codons from TAG to TAA, freeing up TAG to potentially encode a non-standard amino acid," the authors explained. "We also developed a watermark system … allowing facile distinction between synthetic and native genomic content by PCR."

While papers in Cell Genomics and Molecular Cell further explored the 3D consequences of synthetic chromosome fusions or chromosome arm conformation switching, respectively, still other Sc2.0 papers assessed extrachromosomal circular DNA or outlined strategies for quickly generating variant versions of the synthetic sequences or for scaling the incorporation of synthetic chromosomes into individual yeast strains using chromosome substitution.

With the latter chromosome substitution strategy, for example, Zhao, Boeke, and their coauthors put together a yeast strain known as syn7.5 that had synthetic DNA making up more than half of its genome: the 6.5 synthetic chromosomes outlined in their integrated strain, along with another large synthetic chromosome dubbed synIV.

"The international Sc2.0 is a fascinating, highly interdisciplinary project," Daniel Schindler, a researcher affiliated with the Max Planck Institute for Terrestrial Microbiology, the Center for Synthetic Microbiology in Marburg, and the University of Manchester, said in a statement. "It combines basic research to expand our understanding of genome fundamentals, but also paves the way for future applications in biotechnology and drives technology developments."

In a paper published in Cell, co-first author Schindler and colleagues from the UK, Germany, Scotland, and other international centers described a synthetic "neochromosome" that stretches some 190,000 bases and contains sequences for 275 yeast transfer RNAs (tRNAs), which was developed with the help of computer-assisted design and artificial intelligence.

"Our rationale is that removing repetitive elements and relocating tRNA genes onto a dedicated synthetic chromosome will lead to a synthetic yeast genome, which is less susceptible to structural rearrangement and will isolate any instability caused by their presence," members of that team explained.

The tRNA-focused chromosome also points to the possibilities for making further yeast alterations aimed at producing certain supplements, biofuels, or remediation tools.

"As a designer biological structure, the tRNA neochromosome provides opportunities to study how such entities function within the cell," the authors explained, adding that the study "demonstrates the application of these designer structures and the benefits of radically re-engineering the host cell machinery."

"Given rapid technological advances and reduced costs of DNA synthesis," they noted, "we anticipate the routine construction of custom neochromosomes, leading to tangible applications in the near future."