Genome fission and fusion
To further develop the capacities to rearrange genomes at 500-kb to megabase scale, I invented the "genome fission" and "chromosomal fusion" technologies in E. coli 18. In the genome fission operation (Fig. 2a), the single circular wildtype genome is transiently split into two linear genomic fragments (fragment 1 and fragment 2) at defined CRISPR/Cas9 cut sites. Linker 1 contains the luxABCDE operon, while linker 2 has the CmR (positive selection) – sacB (negative selection) double selection cassette and Bacterial Artificial Chromosome (BAC) replication machinery/origin. These linkers are simultaneously excised from the provided "fission BAC". Guided by overlapping homology regions of ~50 bp, the linear genomic fragments are then rejoined with these complementary linker sequences (fragment 1 joined with linker 1, and fragment 2 with linker 2) to form two circular synthetic chromosomes (chromosome 1 and chromosome 2) by lambda-red homologous recombination. One of the synthetic chromosomes is replicated from the original genomic oriC origin while the other is replicated from the synthetic BAC origin18. The fission reaction is driven by the irreversible loss of two copies of a negative selection marker (rpsL) from the system. The size of the secondary chromosome can reach the range of 500-kb to 1-mb scale, limited by the capacity of the BAC origin to stably carry, replicate and segregate with the rearranged genomic fragments.
Following the genome fission reaction, the two synthetic chromosomes of the split genome can be restored back into the original singular circular genomic format by the chromosomal fusion operation (Fig. 2b). Similar to the fission reaction, the fusion reaction is also enabled by cutting open the two synthetic chromosomes using CRISPR/Cas9 and rejoining them into a single circular genome by homologous recombination; this is driven by the irreversible loss of two copies of negative selection marker (pheSmut, one copy per synthetic chromosome) through the process. The two chromosomes can be programmed to fuse in different positions and orientations to generate genome translocations and/or inversions (Fig. 2b)18. I further combined genome fission, chromosome transplantation via conjugation, and chromosomal fusion to assemble genomic regions of 500-kb scale from different E. coli strains into a single genome (Fig. 2c)18.
Fig. 2. Programmed genome fission and chromosomal fusion to enable genome assembly across distinct E. coli strains.