Figure 5. Design flaws at each level of genome complexity.

Genome design flaws can impair fitness or alter the desired functions of an engineered organism. Genome engineering can introduce such design flaws by several mechanisms, including intentional genome changes [39,166], spontaneous mutations [13,148], transposition [165], and genome rearrangements [13]. Point mutations produced during genome engineering can introduce frame shifts, cause amino acid substitutions, de-optimize codon usage, or disrupt the function of overlapping non-coding sequences. Genetic parts such as genes and expression signals can impact crucial cellular functions or the desired function of the engineered organism (e.g., nsAA incorporation, virus resistance, biocontainment). Refactoring genetic pathways provides an opportunity to increase modularity [163], but cryptic regulation mechanisms and polar effects make it difficult to design a de novo pathway architecture with optimal activity [164,180]. Finally, while genome-scale design rules will continue to be discovered, we already know that it is important to balance the size of replichores in circular chromosomes [166], to co-orient transcription of essential operons with translation [168], to preserve sequences involved in DNA structure [169] and repair [170], and to consider how chromosome size impacts its structural integrity [167].