Cre-Lox miRNA-delivery technology optimized for inducible microRNA and gene-silencing studies in zebrafish

Abstract

ABSTRACTWhile many genetic tools exist for zebrafish, this animal model is still lacking a robust gene-silencing and microRNA-delivery technology enabling spatio-temporal control and reliable traceability. We have recently demonstrated that the use of engineered pri-miR backbones can be used to both trigger stable and heritable gene knockdown and/or express microRNA(s) of choice in this organism. This approach/material fills a gap that exists in the zebrafish genetic toolbox to study microRNA biology and has the potential to be a powerful complementary tool to CRISPR-knockout and CRISPR-interference approaches by enabling conditional, traceable tissue-specific and polygenic gene-silencing. However, to date, this miRNA-expressing technology still presents important limitations. First, to trigger potent knockdown(s), multiple synthetic-miRNAs must be expressed simultaneously which compromises the co-expression of fluorescent marker(s) and knockdown traceability, thereby reducing the interest and versatility of this approach. Second, when the gene knockdown triggers significant phenotypes, like homozygous mutants with severe early phenotypes, it is difficult, if not impossible, to maintain adult transgenic carriers. Here, to solve these problems and provide a mature RNAi and microRNA-delivery technology for zebrafish research, we have generated and validated new RNAi reagents and an inducible delivery system based on the Cre/Lox technology. This new system allows the creation of asymptomatic/silent carriers, easing the production of embryos with potent knockdowns that can be traced and spatiotemporally controlled. Following this technological development, we demonstrate the utility of our approach by generating novel inducible models of spinal muscular atrophy (SMA) that open new avenues for studying the normal and pathogenic functions of the smn1 gene, as well as for establishing large-scale screens. Finally, and very importantly, the materials and techniques developed here will simplify research into microRNA biology, as well as conditions involving haploinsufficiency and multiple genes.