High-throughput Experimental Validation of Synonymous Mutation Neutrality in the Human Genome

The assumption that synonymous mutations are fitness-neutral is central to many foundational results in the fields of genetics, genomics, evolutionary biology, and medicine. However, recent results suggest synonymous mutations have pervasive and strong fitness effects. These vigorously debated studies in non-human model systems have even suggested that the proportion of synonymous mutations and their fitness effect sizes are similar to non-synonymous mutations. To systematically test synonymous neutrality across the human genome we utilize recent advances in base editing to probe the fitness effects of 8,558 potential synonymous mutations in 128 highly essential genes. Combined with novel base editing controls and extensive individual sgRNA guide-level validation experiments that quantify false positive rates, we find that synonymous mutations rarely have fitness effects on growth, occurring 165-fold (95% CI, 35-2929 fold) less frequently than non-synonymous fitness-altering mutations. Thus, despite a decade of high-profile controversies, the neutrality assumption for spontaneous synonymous mutations is valid.