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Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterizing the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera , a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, probably a result of non-homologous end joining, and a large deletion in the 3′ untranslated region due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlid radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation. 

Abstract Direct tests of gene function have historically been performed in a limited number of model organisms. The CRISPR/Cas system is species-agnostic, offering the ability to manipulate genes in a range of models, enabling insights into evolution, development, and physiology. Astatotilapia burtoni , a cichlid fish from the rivers and shoreline around Lake Tanganyika, has been extensively studied in the laboratory to understand evolution and the neural control of behavior. Here we develop protocols for the creation of CRISPR-edited cichlids and create a broadly useful mutant line. By manipulating the Tyrosinase gene, which is necessary for eumelanin pigment production, we describe a fast and reliable approach to quantify and optimize gene editing efficiency. Tyrosinase mutants also remove a major obstruction to imaging, enabling visualization of subdermal structures and fluorophores in situ. These protocols will facilitate broad application of CRISPR/Cas9 to studies of cichlids as well as other non-traditional model aquatic species. 

Social hierarchies are ubiquitous in social species and profoundly influence physiology and behavior. Androgens like testosterone have been strongly linked to social status, yet the molecular mechanisms regulating social status are not known. The African cichlid fishAstatotilapia burtoniis a powerful model species for elucidating the role of androgens in social status given their rich social hierarchy and genetic tractability. DominantA. burtonimales possess large testes and bright coloration and perform aggressive and reproductive behaviors while nondominant males do not. Social status inA. burtoniis in flux, however, as males alter their status depending on the social environment. Due to a teleost-specific whole-genome duplication,A. burtonipossess two androgen receptor (AR) paralogs,ARαandARβ, providing a unique opportunity to disentangle the role of gene duplication in the evolution of social systems. Here, we used CRISPR/Cas9 gene editing to generate AR mutantA. burtoniand performed a suite of experiments to interrogate the mechanistic basis of social dominance. We find thatARβ,but notARα, is required for testes growth and bright coloration, whileARα, but notARβ, is required for the performance of reproductive behavior and aggressive displays. Both receptors are required to reduce flees from females and either AR is sufficient for attacking males. Thus, social status inA. burtoniis inordinately dissociable and under the modular control of two AR paralogs. This type of nonredundancy may be important in facilitating social plasticity inA. burtoniand other species whose social status relies on social experience.