Programmed chromosome fission and fusion enable precise large-scale genome rearrangement and assembly

by Loriejoice Anne Manalo (Polyplex)

A study conducted on August 30, 2019 explored the application of chromosome fission and fusion on Escherichia coli for creating synthetic genomes. There are already existing methods for synthetic genome design of E. coli such as iterative replacement of genomic DNA with synthetic DNA, deletion of genomic DNA, translocation and inversion of large genomic sections, and combining large genome sections from distinct strains, or the convergent assembly of synthetic genomes. However, these methods have certain limitations because they require large regions of homology, and since the crossover site between the two genomes is not precisely specified, undesired chimeras may occur.

Chromosome fission and fusion occurs in natural evolution and may even have helped in accelerating evolution itself. In this study, it was demonstrated that the E. coli genome can be efficiently split by single-step programmed fission into pairs of synthetic chromosomes. This resulted to chromosomes that enable precise programmed fusions, genomic inversions, and translocations. It also provided a route to assemble new genomes through the precise, convergent assembly of large genomic fragments from distinct strains.

The researchers designed and synthesized a system that will precisely split the unmodified genome of E. coli into two user-defined, circular chromosomes. The E. coli genome was split into two chromosomes and was further cut into fragments and linker sequences by introducing Cas9 with appropriate spacers, lambda-red recombination machinery, and fission bacterial artificial chromosomes (BAC) into cells.

Successful fission resulted to streptomycin-resistant and selectively sucrose-sensitive cells. It was then enriched to generate two chromosomes through growth on streptomycin and chloramphenicol. This selects for both loss of rpsL and maintenance of CmR in the SacB-CmR double selection cassette and therefore kills cells containing the fission BAC but allows growth of cells that have undergone programmed genome fission.

Fission had only modest effects on the growth of cells. Genome fissions were present after approximately 105 generations of continuous growth based from the researchers’ observations. Programmed fusion of synthetic chromosomes, generated by FISSION, demonstrated that it enables the generation of precisely rearranged genomes.

The study did not only use fission and fusion. The researchers also utilized conjugative transplantation to precisely combine defined sections of distinct genomes. This resulted to successful attempts for all genome assemblies. They also demonstrated the efficiently programmed, single-step fission of the unmodified E. coli genome into diverse megabase-scale chromosomes. Chromosomes in a single cell can be fused into a single genome to effect precise genomic translocations or precise and scarless inversions.

The realization of reorganized genome designs and the exploration of modular, synthetic syntenies may have applications to engineering. The processes done enable the new genomes to precisely converge. Also, their work provides the necessary set of precise, rapid, large-scale genome-engineering operations for creating diverse synthetic genomes.

View full text here: https://science.sciencemag.org/content/365/6456/922