A robust and flexible CRISPR/Cas9-based system for neutrophil-specific gene inactivation in zebrafish.

Journal Article (Journal Article)

CRISPR/Cas9-based tissue-specific knockout techniques are essential for 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 single guide (sg)RNAs 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 genes restores 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.

Full Text

Duke Authors

Cited Authors

  • Wang, Y; Hsu, AY; Walton, EM; Park, SJ; Syahirah, R; Wang, T; Zhou, W; Ding, C; Lemke, AP; Zhang, G; Tobin, DM; Deng, Q

Published Date

  • April 15, 2021

Published In

Volume / Issue

  • 134 / 8

PubMed ID

  • 33722979

Pubmed Central ID

  • PMC8084575

Electronic International Standard Serial Number (EISSN)

  • 1477-9137

Digital Object Identifier (DOI)

  • 10.1242/jcs.258574

Language

  • eng

Conference Location

  • England