Synonymous edits in the <i>Escherichia coli</i> genome have substantial and condition-dependent effects on fitness

CRISPR-Cas-based genome editing is widely used in bacteria at scales ranging from construction of individual mutants to massively parallel libraries. This procedure relies on guide RNA-directed cleavage of the genome followed by repair with a template that introduces a desired mutation along with synonymous "immunizing" mutations to prevent re-cleavage of the genome after editing. Because the immunizing mutations do not change the protein sequence, they are often assumed to be neutral. However, synonymous mutations can change mRNA structures in ways that alter levels of the encoded proteins. We have tested the assumption that immunizing mutations are neutral by constructing a library of over 50,000 edits that consist of only synonymous mutations in <i>Escherichia coli</i>. Thousands of edits had substantial effects on fitness during growth of <i>E. coli</i> on acetate, a poor carbon source that is toxic at high concentrations. The percentage of high-impact edits varied considerably between genes and at different positions within genes. We reconstructed clones with high-impact edits and found that 69% indeed had significant effects on growth in acetate. Interestingly, fewer edits affected fitness during growth in glucose, a preferred carbon source, suggesting that changes in protein expression caused by synonymous mutations may be most important when an organism encounters challenging conditions. Finally, we showed that synonymous edits can have widespread effects; a synonymous edit at the 5' end of <i>ptsI</i> altered expression of hundreds of genes. Our results suggest that the synonymous immunizing edits introduced during CRISPR-Cas-based genome editing should not be assumed to be innocuous.