ZFIN ID: ZDB-PUB-210317-7
A robust and flexible CRISPR/Cas9 based system for neutrophil-specific gene inactivation in zebrafish
Wang, Y., Hsu, A.Y., Walton, E.M., Park, S.J., Syahirah, R., Wang, T., Zhou, W., Ding, C., Lemke, A.P., Zhang, G., Tobin, D.M., Deng, Q.
Date: 2021
Source: Journal of Cell Science   134(8): (Journal)
Registered Authors: Deng, Qing, Hsu, Alan, Syahirah Mohd Sabri, Ramizah, Tobin, David, Wang, Tianqi, Wang, Yueyang, Zhang, GuangJun, Zhou, Wenqing
Keywords: Actin stress, Cell migration, Leukocytes, Live imaging, Rac2
MeSH Terms:
  • Animals
  • Animals, Genetically Modified
  • CRISPR-Cas Systems/genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats/genetics
  • Neutrophils*/metabolism
  • Zebrafish*/genetics
  • Zebrafish*/metabolism
  • Zebrafish Proteins/genetics
  • Zebrafish Proteins/metabolism
  • rac GTP-Binding Proteins/metabolism
PubMed: 33722979 Full text @ J. Cell Sci.
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.