Our lab at Imperial, in collaboration with Ben Blount at University of Nottingham and many others finally completed construction of Syn11, a synthetic yeast chromosome, as part of the major international Sc2.0 project to build the world’s first synthetic yeast genome.
The work, which we published in Cell Genomics in Novemeber 2023, represents completion of one of the 16 chromosomes of the yeast genome by our lab over a 10 year period. The international collaboration, known as 'Sc2.0' has been a 15-year project involving teams from around the world (UK, US, China, Singapore, UK, France and Australia), working together to make synthetic versions of all of yeast's chromosomes. Alongside our paper, another 9 publications were also released on the same day from other teams describing their synthetic chromosomes. The final completion of the genome project - the largest synthetic genome ever - is expected by 2025.
Our synthetic chromosome, over 650 kb in length has 1000s of synonmous mutations in coding genes but these cause no issues at all. However in several places edits to non-coding DNA did cause problems. Fixing these required a painstaking debugging process. In particular we saw issues based on deletion and insertion of sequence in non-coding DNA close to key features - genes encoding mitocondrial proteins, the centromere. Unlike what some recent work might say, we see no evidence of any fitness loss due to synonmous mutations.
We also saw issues that arose from DNA assembly, including one chunk of DNA that inserted itself many many times, giving us a chromosome >100 kb longer than we wanted. Some fancy CRISPR work by Ben Blount managed to chop that all out in a perfect way. One design choice of Sc2.0 is to remove all transposons from the yeast genome, so it was ironic that our assembly method inserted a bacterial transposable element into one of the yeast genes. This must've jumped-in during plasmid prep for the synthetic DNA chunk. Illumina sequencing and alignment of reads to a reference never revealed this, as we never expected bacterial DNA to be present. We only spotted this thanks to Nanopore sequencing.
Rather than being a straight copy of the natural genome, the Sc2.0 synthetic genome has been designed with new features that give cells novel abilities not found in nature. One of these features allows researchers to force the cells to shuffle their gene content, creating millions of different versions of the cells with different characteristics. Individuals can then be picked with improved properties for a wide range of applications in medicine, bioenergy and biotechnology. The process is effectively a form of super-charged evolution.
In our paper we show that the design of SynXI can be repurposed to provide a new system to study a specific type of structural rearrangment that occurs in genomes - that of extrachromosomal circular DNAs (eccDNAs). These are free-floating DNA circles that have “looped out” of the genome and are being increasingly recognised as factors in ageing and as a cause of malignant growth and chemotherapeutic drug resistance in many cancers, including glioblastoma brain tumours.
The synthetic chromosomes are massive technical achievements in their own right, but they can also open up a huge range of new abilities for how we study and apply biology. This could range from creating new microbial strains for greener bioproduction, through to helping us understand and combat disease. And furthermore, by constructing a redesigned chromosome from telomere to telomere, and showing it can replace a natural chromosome just fine, this research establishes the foundations for designing and making synthetic chromosomes and even genomes for complex organisms like plants and animals
For this work we collaborated with the universities of Edinburgh, Cambridge, and Manchester in the UK, as well as John Hopkins University and New York University Langone Health in the USA and Universidad Nacional Autónoma de México, Querétaro in Mexico.
The full paper can be found here and the work was funded by the BBSRC.
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