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

Abstract

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.

A fresh look at mycobacterial pathogenicity with the zebrafish host model

“…Further use of the different mycobacterial infection models would benefit from expanding the toolbox for precision genetic engineering and the collection of fluorescent marker lines. Cell‐specific gene inactivation has long been a bottleneck but has now been achieved for both neutrophils and macrophages (Isiaku et al., 2021 ; Wang et al., 2021 ), and this technology provides great opportunities to analyze gene functions in a lineage‐specific manner. Proinflammatory responses in macrophages can be visualized using tnfa:gfp and ilb:gfp transgenic lines (Lewis & Elks, 2019 ; Ogryzko et al., 2019 ), but the tools are still lacking to detect other polarization states that make monocytes and macrophages permissive to mycobacterial growth.…”

“…Cell-specific gene inactivation has long been a bottleneck but has now been achieved for both neutrophils and macrophages (Isiaku et al, 2021;Wang et al, 2021), and this technology provides great opportunities to analyze gene functions in a lineage-specific manner.…”

Progress in Gene-Editing Technology of Zebrafish

“…In 2021, Isiaku and his colleagues [ 62 ] coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages ( Table 1 ). They injected two gRNAs targeting trim33 into Tg (mpx-Cas9) and Tg (mpeg1-Cas9) lines.…”

Shifting the focus of zebrafish toward a model of the tumor microenvironment

“…Similarly, the MASERATI plasmid has been used to knockout genes specifically in melanocytes to generate melanomas ( Ablain et al, 2018 ). Another transient approach based on the injection of chemically modified synthetic gRNAs into embryos of transgenic fish expressing Cas9 in a lineage-specific matter based on the GAL4/UAS system showed promising results in gene editing of zebrafish neutrophils and macrophages and could be in principle extended to other cell types of interest ( Isiaku et al, 2021 ). With many newer CRISPR systems now available one can perform unique genetic edits in different TME cell types simultaneously ( Liu et al, 2019 ).…”