Fig. 4: Multiplex gene deletion with CReATiNG.

a We attempted to delete 10 non-adjacent regions of the chromosome core and both subtelomeres from BY ChrI, totaling 39.9% of the chromosome. To do this, we cloned 11 segments of the ChrI, assembled them, and performed native chromosome elimination. b ONT sequencing of a colony confirmed results from PCR checks. The colony with the most deletions (nine regions) had the correct structure, but had retained the region containing SYN8. An in silico design of the chromosome is shown with a subset of mapped reads plotted below it. c Growth rate analysis of different BY strains, including the unaltered reference strain (BY), a strain carrying a synthetic circular ChrI lacking subtelomeres (BY synthetic ChrI), a synthetic circular ChrI lacking nine core regions and both subtelomeres (multiple deletion ChrI), a synthetic circular ChrI lacking nine core regions, both subtelomeres, and SYN8 (multiple deletion ChrI syn8∆), and the reference strain with SYN8 deleted (BY syn8∆). For each strain, a total of nine independent growth experiments were performed (n = 9). Each dot represents the value from an individual replicate of a given genotype, while the horizontal bar represents the mean across the nine replicates. d Recombination between synthetic and native copies of ChrI produced synthetic chromosomes with SYN8.