Genome Transplantation 1 in Mollicutes

Genome transplantation (GT) is a technique enabling the installation and boot up of a marked natural or synthetic donor chromosome into a recipient cytoplasm. The donor genomes can be assembled and modified in the yeast Saccharomyces cerevisiae, which can stably maintain chromosomes with a size of more than 1 Mbp as yeast artificial chromosomes. The use of S. cerevisiae as the intermediary host of the donor chromosome allows the manipulation of the prokaryotic chromosome before transplantation via techniques such as tandem repeat coupled with endonuclease cleavage (TREC), TREC-assisted gene knock-in (TREC-IN), CRISPR/Cas9 editing or transformation-associated recombination (TAR) cloning. The chromosomes need to be transplanted intact, which can be achieved by trapping cells in low melting agarose plugs followed by the release of the chromosomes after lysis of the cells and complete digestion of proteins. GT is facilitated by cell fusion enabled by polyethylene glycol.
As a result of GT, the original chromosome is replaced 23 by the donor chromosome which becomes the new operating system of the recipient cell. Mollicutes were first used for GT due to their small-sized chromosomes and absence of bacterial cell wall, which likely facilitates this process. GT has been successfully adapted to several members of the ‘Spiroplasma phylogenetic group’ of Mollicutes, including well-known livestock pathogens. Several limiting factors, including the presence of restriction-methylation systems in the recipient cell and the phylogenetic distance between recipient and donor cells impacts the success rate of GT. Overall, GT is a technique with great prospects in the field of synthetic biology, which paved the way for the creation of the first synthetic cell, although its adaptation to other species of the Mollicutes class and beyond is still pending to fully unlock its potential.