Recapitulating evolutionary divergence in a singlecis-regulatory element is sufficient to cause expression changes of the lens geneTdrd7

Abstract

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved non-coding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated the evolutionary changes by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor element has a large effect, causing a more than two-fold up-regulation of Tdrd7 in developing lens. Interestingly, the increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, likely explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eyeregulatory elements collectively contribute to phenotypic differences.3

Convergent and lineage-specific genomic differences in limb regulatory elements in limbless reptile lineages

“…Third, experiments are required to narrow the list of candidates to those that have the potential to directly affect the trait of interest. Ideally, such experiments precisely recapitulate the naturally occurring mutations in a model organism possessing the trait, which is possible by swapping the sequence of regulatory elements with CRISPR-Cas9 (Kvon et al, 2016;Roscito et al, 2021). However, such model organism experiments are hard to scale up to test a larger number of promising regulatory elements.…”