RT Journal Article
SR Electronic
T1 A CRISPR/Cas9 vector system for neutrophil-specific gene disruption in zebrafish
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.07.27.223008
DO 10.1101/2020.07.27.223008
A1 Yueyang Wang
A1 Alan Y. Hsu
A1 Eric M. Walton
A1 Ramizah Syahirah
A1 Tianqi Wang
A1 Wenqing Zhou
A1 Chang Ding
A1 Abby Pei Lemke
A1 David M. Tobin
A1 Qing Deng
YR 2020
UL http://biorxiv.org/content/early/2020/07/28/2020.07.27.223008.abstract
AB Tissue-specific knockout techniques are widely applied in biological studies to probe the tissue-specific roles of specific genes in physiology, development, and disease. CRISPR/Cas9 is a widely used technology to perform fast and efficient genome editing in vitro and in vivo. Here, we report a robust CRISPR-based gateway system for tissue-specific gene inactivation in zebrafish. A transgenic fish line expressing Cas9 under the control of a neutrophil-restricted promoter was constructed. As proof of principle, we transiently disrupted rac2 or cdk2 in neutrophils using plasmids driving the expression of sgRNAs from U6 promoters. Loss of the rac2 or cdk2 gene in neutrophils resulted in significantly decreased cell motility, which could be restored by re-expressing Rac2 or Cdk2 in neutrophils in the corresponding knockout background. The subcellular location of Rac activation and actin structure and stress in the context of neutrophil migration was determined in both the wild-type and rac2 knockout neutrophils in vivo. In addition, we evaluated an alternative approach where the Cas9 protein is ubiquitously expressed while the sgRNA is processed by ribozymes and expressed in a neutrophil-restricted manner. Cell motility was also reduced upon rac2 sgRNA expression. Together, our work provides a potent tool that can be used to advance the utility of zebrafish in identification and characterization of gene functions in neutrophils.Competing Interest StatementThe authors have declared no competing interest.