Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions
- Isiaku, A.I., Zhang, Z., Pazhakh, V., Manley, H.R., Thompson, E.R., Fox, L.C., Yerneni, S., Blombery, P., Lieschke, G.J.
- Disease models & mechanisms 14(7): (Journal)
- Registered Authors
- Manley, Harriet, Pazhakh, Vahid
- CRISPR-Cas9, Cell autonomy, Gene editing, Macrophages, Neutrophils, Zebrafish
- MeSH Terms
- Animals, Genetically Modified
- CRISPR-Cas Systems/genetics
- Gene Editing*
- Transcription Factors/metabolism
- 34296745 Full text @ Dis. Model. Mech.
Isiaku, A.I., Zhang, Z., Pazhakh, V., Manley, H.R., Thompson, E.R., Fox, L.C., Yerneni, S., Blombery, P., Lieschke, G.J. (2021) Transient, flexible gene editing in zebrafish neutrophils and macrophages for determination of cell-autonomous functions. Disease models & mechanisms. 14(7):.
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.
Genes / Markers
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes