High throughput editing screen provide direct experimental validation of synonymous mutation neutrality in the human genome
Assuming synonymous mutations are mostly 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. Those vigorously debated studies in non-human model systems have even suggested that the proportion of synonymous mutations and their fitness effect size is similar to non-synonymous mutations. To systematically test synonymous neutrality across the human genome we utilize a recently developed functional genomic technique known as base editing to probe the fitness effects of 8,558 potential synonymous mutations in 128 human 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 at a rate one-hundred fold smaller than for non-synonymous mutations. Thus, the neutrality assumption of synonymous mutations is valid when single mutations are introduced into a gene.