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

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-...

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Bibliographic Details
Published in:Disease models & mechanisms 2021-07, Vol.14 (7)
Main Authors: Isiaku, Abdulsalam I, Zhang, Zuobing, Pazhakh, Vahid, Manley, Harriet R, Thompson, Ella R, Fox, Lucy C, Yerneni, Satwica, Blombery, Piers, Lieschke, Graham J
Format: Article
Language:English
Subjects:
Animals
Animals, Genetically Modified
cell autonomy
CRISPR-Cas Systems - genetics
crispr-cas9
Gene Editing
Gene expression
Genome editing
Heart
Humans
Localization
macrophages
Macrophages - metabolism
Neutrophils
Neutrophils - metabolism
Physiology
Resource
Transcription Factors - metabolism
zebrafish
Zebrafish - genetics
Zebrafish - metabolism
Zebrafish as a Disease Model
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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.
ISSN:1754-8403
1754-8411
DOI:10.1242/dmm.047431