Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions

• Main Authors: Abdulsalam I. Isiaku (Author), Zuobing Zhang (Author), Vahid Pazhakh (Author), Harriet R. Manley (Author), Ella R. Thompson (Author), Lucy C. Fox (Author), Satwica Yerneni (Author), Piers Blombery (Author), Graham J. Lieschke (Author)
• Format: Book
• Published: The Company of Biologists, 2021-07-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.

Summary

Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing individual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.

Item Description

1754-8403
1754-8411
10.1242/dmm.047431