CRISPR/Cas9-based tissue-specific knockout techniques are essential in probing the functions of genes in embryonic development and disease using zebrafish. However, the lack of capacity to perform gene-specific rescue or live-imaging in the tissue-specific knockout background has limited the utility of this approach. Here, we report a robust and flexible gateway system for tissue-specific gene inactivation in neutrophils. Using a transgenic fish line with neutrophil-restricted expression of Cas9 and ubiquitous expression of sgRNAs targeting rac2, specific disruption of the rac2 gene in neutrophils is achieved. Transient expression of sgRNAs targeting rac2 or cdk2 in the neutrophil-restricted Cas9 line also results in significantly decreased cell motility. Re-expressing sgRNA-resistant rac2 or cdk2 gene restored neutrophil motility in the corresponding knockout background. Moreover, active Rac and force bearing F-actins localize to both the cell front and the contracting tail during neutrophil interstitial migration in an oscillating fashion that is disrupted when rac2 is knocked out. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identifying and characterizing gene functions in a tissue-specific manner.

Background Inwardly rectifying potassium channels are essential for normal potassium homeostasis, maintaining the cellular resting membrane potential, and regulating electrolyte transportation. Mutations in Kir channels have been known to cause debilitating diseases ranging from neurological abnormalities to renal and cardiac failures. Many efforts have been made to understand their protein structures, physiological functions, and pharmacological modifiers. However, their expression and functions during embryonic development remain largely unknown. Results Using zebrafish as a model, we identified and renamed 31 kir genes. We also analyzed Kir gene evolution by phylogenetic and syntenic analyses. Our data indicated that the four subtypes of the Kir genes might have already evolved out in chordates. These vertebrate Kir genes most likely resulted from both whole‐genome duplications and tandem duplications. In addition, we examined zebrafish kir gene expression during early embryogenesis. Each subgroup's genes showed similar but distinct gene expression domains. The gene expression of ohnologous genes from teleost‐specific whole‐genome duplication indicated subfunctionalization. Varied temporal gene expression domains suggest that Kir channels may be needed for embryonic patterning or regulation. Conclusions Our phylogenetic and developmental analyses of Kir channels shed light on their evolutionary history and potential functions during embryogenesis related to congenital diseases and human channelopathies.