The eukaryotic cell cycle, a pivotal biological process, has been extensively studied and
mathematically modelled in recent decades. Despite concerted efforts, identifying the minimal gene set essential for orderly cell cycle progression remains elusive. Synthetic biology, renowned for genetic engineering applications, also provides a pathway for addressing fundamental biological queries through “learning from building.” The Synthetic Yeast Genome (Sc2.0) project exemplifies this by synthesising Saccharomyces cerevisiae’s genome with changes that advance our understanding of eukaryotic genomes.
Expanding from Sc2.0’s groundwork, we aim to pioneer synthetic yeast genomes that are
minimal, modular, and reprogrammable. As a proof-of-concept, we constructed a synthetic
genome module housing nine of the key cell cycle genes. Employing CRISPR, we
systematically deleted these genes from their native loci and reinserted them together as a
synthetic gene cluster. While individually non-essential, the combined absence of all nine
genes renders this synthetic module indispensable.
Through Cre/loxP-mediated recombination, we investigated the gene combinations necessary for yeast cell cycle progression. Cre recombinase facilitated targeted gene deletions between intergenic loxP sites within the module, and rapidly generated diverse strains with combinatorial cluster deletion profiles, covering all potential combinations. Using flow cytometry sorting, we developed a way to isolate hundreds of viable deletion combinations and developed the Pool of Long Amplified Reads (POLAR) sequencing technique to enable the analysis of gene deletion frequency and gene content combinations for hundreds of strains with different cell cycle modules. These experimental findings were compared to computational models of the cell cycle and get us closer to understanding the minimal gene content for this function.
Upon pioneering this work, we now envisage a future where genome designers can predict
gene sets necessary for specialised tasks and can then synthetically arrange these genes on chromosomes and design intergenic regions to regulate their gene expression appropriately.