Zebrafish as a Model for Evaluating Coilin‐Mediated microRNA Biogenesis

Abstract

Cajal bodies (CBs) are nuclear bodies that are hosts for the modification and assembly of ribonucleoproteins (RNPs). The nuclear protein coilin is known as the CB marker protein as it is the scaffold for CB assembly and vital for structural integrity. However, there is still not much understood about the function of coilin. Recent studies by our lab implicate coilin in the biogenesis of microRNAs (miRNAs). Currently, there are sparse options in animal models for studying coilin and CB function that account for limitations such as off‐target effects of coilin depletion or incomplete protein knockdown. Due to this, we sought to develop novel coilin mutations in zebrafish to advance functional studies of this protein and CBs. In considering how we would approach this, we opted for the innovative CRISPR/Cas9 technology as an optimal strategy and hypothesized that the implementation of this genome editing technology would allow us to develop multiple lines of coilin‐modified zebrafish for studies investigating coilin’s role in miRNA biogenesis. We selected a suitable Protospacer Adjacent Motif (PAM) sequence and designed guide RNAs for our mutations of interest in silico, which we then generated in vitro, and injected into fertilized zebrafish embryos at the one‐cell stage. An initial screening for insertions or deletions (indels) took place at 72 hours post fertilization to determine mutation frequency and once again when the zebrafish reached adulthood. Through our efforts, we have managed to obtain zebrafish that are heterozygous for coilin‐null mutations and are currently in the midst of screening for additional lines of coilin mutations which could serve to characterize the functional domains of coilin in vivo. Utilizing CRISPR/Cas9 technology, we have generated the first coilin‐null mutation in a zebrafish line allowing our lab to expand our investigations into the role of coilin and CBs in the regulatory network of the nucleus.