Fig. 1

Design principle of the CRISPR-based chromosomal-separation technique developed in this study. A The editing cassettes 1 and 2 are inserted into the chromosome by homologous recombination, forming the E. coli^C1/C2 strain. Subsequently, the CRISPR/Cas9 system is expressed to generate two double-strand breaks (DSBs) at two inserted N20PAM sequences on the editing cassettes 1 and 2, respectively. As designed, the two halves of the replication origin combine into a complete origin by recombination and form a Chr.0.10 M, while the Chr.4.54 M containing the original replication origin is constructed from the rest of the chromosome. Thus, the E. coli^0.10/4.54 strain was created using this CRISPR-based chromosomal-separation technique. B To obtain a strain bearing two large chromosomes, the genome engineering strategy was redesigned to implement a selection pressure for cells carrying the newly created chromosomes. An ampicillin resistant gene, amp^r, was separated into two halves and inserted into the chromosome along with the split E. coli origin of replication. Thus, after CRISPR-induced chromosomal DNA recombination, a complete amp^r gene was pieced together from the halves along with a second origin of replication that was also newly formed