Tissue-specific gene targeting using CRISPR/Cas9

Abstract

The zebrafish has been a powerful model in forward genetic screens to identify genes essential for organogenesis and embryonic development. Conversely, using reverse genetics to investigate specific gene function requires phenotypic analysis of complete gene inactivation. Despite the availability and efficacy of morpholinos, the lack of tractable and efficient knockout technologies has impeded reverse genetic studies in the zebrafish, particularly in adult animals. The recent development of genome-editing technologies such as CRISPR/Cas9 greatly widened the scope of loss-of-function studies in the zebrafish, allowing for the rapid phenotypic assessment of gene silencing in embryos, the generation of knockout lines, and large-scale reverse genetic screens. Tissue-specific gene inactivation would be ideal for these studies given the caveats of whole-embryo gene silencing, yet spatial control of gene targeting remains a challenge. In this chapter, we focus on tissue-specific gene inactivation using the CRISPR/Cas9 technology. We first explain the rationale for this technique, including some of its potential applications to tackle important biological issues and the inability of current technologies to address these issues. We then present a method to target genes in a tissue-specific manner in the zebrafish. Finally, we discuss technical difficulties and limitations of this method as well as possible future developments.

3D-printable tools for developmental biology: Improving embryo injection and screening techniques through 3D-printing technology

“…In order to successfully accomplish a comprehensive project, lineage tracing and transgenesis experiments are often performed. These studies generally require the injection of reagents such as dextran, diI, morpholinos, RNAi, mRNA in vitro synthesized or constructs inside vectors [3,4,5,6]. Subsequently, checking the embryos, or the frequently highly mobile juveniles, is an essential next step for correctly describing a phenotype or a whole in vivo process [7,8].…”

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

“…Consistent with our MO findings, fzd9b mutants had a reduction in cmyb+ cells at 40 hpf ( Fig 2H-I). Finally, we used a previously described transgenic approach where a guide RNA to fzd9b is expressed ubiquitously and Cas9 expression is spatially regulated by UAS to conditionally inactivate fzd9b in early endothelial cells (Ablain and Zon, 2016), which also led to a reduction in the number of cmyb+ cells at 40 hpf , indicating that Fzd9b is required in the endothelium for HSPC development. Taken together, these results indicate Fzd9b is required for HSPC development, downstream of fate specification, and specifically in endothelial cells.…”

Quo natas, Danio?—Recent Progress in Modeling Cancer in Zebrafish

“…Frequent bi-allelic targeting observed with CRISPR/Cas9 saves time spent back-crossing fish lines to homozygosity. Cell type-specific knockouts can be achieved by expression of Cas9 under a tissue-specific promoter allowing for spatial control of gene disruption in somatic cells ( 31 ). Although still a challenge, progress has been made in establishing knockin strategies targeting an endogenous cancer-relevant locus by homologous recombination ( 32 – 34 ).…”