How to build a genome

Leading synthetic biologists have shared hard-won lessons from their decade-long quest to build the world's first synthetic eukaryotic genome in a Nature Biotechnology paper out today. Their insights could accelerate development of the next generation of engineered organisms, from climate-resilient crops to custom-built cell factories.

"We've assembled a comprehensive overview of the literature on how to build a life form where we review what went right – but also what went wrong," says Dr Paige Erpf, lead author of the paper and postdoctoral researcher at Macquarie University's School of Natural Sciences and the Australian Research Council (ARC) Centre of Excellence in Synthetic Biology.

The Synthetic Yeast Genome Project (Sc2.0) involved a large, evolving global consortium of 200-plus researchers from more than ten institutions, who jointly set out to redesign and chemically synthesise all 16 chromosomes of baker's yeast from scratch. Macquarie University contributed to the synthesis of two of these chromosomes, comprising around 12 per cent of the project overall.

The process for each chromosome followed the same design principles: removing unstable genetic elements; introducing molecular 'watermarks' to distinguish synthetic DNA from natural sequences; and adding the gene-shuffling system 'SCRaMbLE' so researchers could rearrange genes and test their functions.

Rewriting the blueprint

Unlike traditional genetic engineering, which tweaks existing genomes, Sc2.0 was the first to rewrite an entire genome from the ground up – all 12 million base pairs of it.

"Completing all 16 synthetic chromosomes lets us understand genome function at a scale that was simply impossible before," says Distinguished Professor Ian Paulsen, director of the ARC Centre of Excellence in Synthetic Biology at Macquarie.

The chromosomes were assembled in large chunks containing thousands of base pairs, then integrated into living yeast cells step by step, relying on yeast's own cellular machinery to stitch the synthetic pieces into place.

Learning from mistakes

Despite the standardised design principles, every research team encountered similar problems. The paper catalogues these 'bugs' systematically, offering future synthetic biologists a roadmap of what to avoid.

Tiny DNA watermarks, designed to be silent, occasionally disrupted gene function in unexpected ways. Some genes flagged as non-essential turned out to cause significant growth problems when removed.

Yeast cannot regenerate mitochondrial genomes from scratch, so any damage required researchers to perform a genetic rescue operation, where they identified and fixed the problem, then had to reintroduce healthy mitochondria through careful breeding.

Teams developed and shared sophisticated debugging tools, such as 'Pooled PCRtag Mapping' (which allows researchers to screen hundreds of yeast colonies simultaneously to pinpoint which genetic changes caused problems) and 'CRISPR D-BUGS' (combines gene editing with selection strategies).

Dr Hugh Goold led work on synXVI, one of Macquarie's synthetic chromosomes, for the NSW Department of Primary Industries and Regional Development.

"The hardest challenges were both psychological and technical: the long haul of a decade-long project where progress could feel painfully slow, and the difficulty of working with cells that were unfit and difficult to grow," says Dr Goold.

Next-generation crops

The lessons from yeast are already informing bold new projects. A Macquarie research team led by Dr Briardo Lorente and Distinguished Professor Ian Paulsen from the Australian Genome Foundry recently began work on building the world's first synthetic crop chromosome, part of a AUD $24 million / £12 million UK-funded effort to develop the technology to create synthetic plant genomes.

Plants grow slowly and are far more difficult to engineer than yeast, so this project uses an ingenious approach: building the synthetic plant chromosomes inside yeast cells first, then transferring the newly constructed chromosome into plant cells.

"The 'learning by building' approach taken by the Sc2.0 consortium gave us incredible insights into genetics that we may not have fully grasped had we taken historical step-by-step approaches," Dr Goold says.

But the biggest lessons from this project may still be to come. "Putting all the synthetic DNA into a single cell will be a momentous occasion and will lead to even deeper understanding of the biology that underpins our environment, our food systems and our medicines," says Dr Goold.