SRP309850 PRJNA706779 WideSeq of Zebrafish rac2 exon 8 locus after CRISPR knockout 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 in the neutrophil-restricted Cas9 line also results in significantly decreased cell motility. Re-expressing sgRNA-resistant rac2 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 are 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. Danio rerio strain:AB