The challenge of dissecting gene function in model organisms: Tools to characterize genetic mutants and assess transcriptional adaptation in zebrafish

Genome editing technologies including the CRISPR/Cas9 system have greatly improved our knowledge of gene function and biological processes, however, these approaches have also brought new challenges to determining genotype-phenotype correlations. In this chapter, we briefly review gene-editing technologies used in zebrafish and discuss the differences in phenotypes that can arise when gene expression is inhibited by anti-sense or by gene editing techniques. We outline possible explanations for why knockout phenotypes are milder, tissue-restricted, or even absent, compared with severe knockdown phenotypes. One proposed explanation is transcriptional adaptation, a form of genetic robustness that is induced by deleterious mutations but not gene knockdowns. Although much is unknown about what triggers this process, its relevance in shaping genome expression has been shown in multiple animal models. We recently explored if transcriptional adaptation could explain genotype-phenotype discrepancies seen between two zebrafish models of the centrosomal protein Cep290 deficiency. We compared cilia-related phenotypes in knockdown (anti-sense) and knockout (mutation) Cep290 models and showed that only cep290 gene mutation induces the upregulation of genes encoding the cilia-associated small GTPases Arl3, Arl13b, and Unc119b. Importantly, the ectopic expression of Arl3, Arl13b, and Unc119b in cep290 morphant zebrafish embryos rescued cilia defects. Here we provide protocols and experimental approaches that can be used to explore if transcriptional adaptation may be modulating gene expression in a zebrafish ciliary mutant model.