CRISPR screening reveals genetic regulators associated with the evolution of eye degeneration

Determining the genetic factors contributing to trait evolution is critical for understanding how and why traits evolve; however, establishing which genes underlie the evolution of complex traits remains challenging. The freshwater fish <i>Astyanax mexicanus</i>, a species that includes blind, cave-dwelling and eyed, surface-dwelling fish, is a powerful model for evolutionary genetics. While genetic mapping studies in this species previously identified genomic regions associated with cave-derived traits, few causative genes and genetic changes have been identified. Here, we develop methods to identify and rapidly functionally assess candidate genes in <i>A. mexicanus</i>, focusing on a defining trait of cave animals, eye loss. Candidate genes were identified based on whether they fell within an eye-related quantitative trait locus, were differentially expressed between surface and cave eyes, and showed evidence of positive selection in cavefish. Single-nucleus RNA-sequencing revealed that these candidate genes were expressed in multiple cell types during development, including those in different tissues of the eye. CRISPR-Cas9-based mutagenesis demonstrated that disruption of nine of these candidate genes in surface fish resulted in altered eye size. Perturbation of one of these genes, <i>fibulin-7</i> (<i>fbln7)</i>, revealed changes in eye size across multiple stages of eye development. Together, this work identified multiple genes associated with the evolution of eye degeneration in <i>A. mexicanus</i>. Further, this study represents a roadmap for rapid identification and functional assessment of candidate genes implicated in the evolution of traits in cavefish that can be applied to other evolutionary genetic models.