A CRISPR-based chromosomal-separation technique for Escherichia coli

BACKGROUND: Natural life systems can be significantly modified at the genomic scale by human intervention, demonstrating the great innovation capacity of genome engineering. Large epi-chromosomal DNA structures were established in Escherichia coli cells, but some of these methods were inconvenient, using heterologous systems, or relied on engineered E. coli strains. RESULTS: The wild-type model bacterium E. coli has a single circular chromosome. In this work, a novel method was developed to split the original chromosome of wild-type E. coli. With this method, novel E. coli strains containing two chromosomes of 0.10 Mb and 4.54 Mb, and 2.28 Mb and 2.36 Mb were created respectively, designated as E. coli(0.10/4.54) and E. coli(2.28/2.36). The new chromosomal arrangement was proved by PCR amplification of joint regions as well as a combination of Nanopore and Illumina sequencing analysis. While E. coli(0.10/4.54) was quite stable, the two chromosomes of E. coli(2.28/2.36) population recombined into a new chromosome (Chr.4.64M(Mut)), via recombination. Both engineered strains grew slightly slower than the wild-type, and their cell shapes were obviously elongated. CONCLUSION: Finally, we successfully developed a simple CRISPR-based genome engineering technique for the construction of multi-chromosomal E. coli strains with no heterologous genetic parts. This technique might be applied to other prokaryotes for synthetic biology studies and applications in the future. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-022-01957-4.