Targeted RNA knockdown by crRNA guided Csm in zebrafish

Abstract

RNA interference (RNAi) is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a \"proof of principle\" demonstration that CRISPR endoribonucleases can be used for programmable mRNA transcript degradation. Using zebrafish as the animal model and Csm(crRNA) complexes as the CRISPR endoribonucleases, we have targeted a transgenic EGFP transcript expressed from a variety of promoters. A drastic decrease of fluorescence was achieved in germ cells of the vasa:EGFP line. Weaker effects were also seen in fish lines that express EGFP zygotically. Knockdown was statistically significant in cmcl2:EGFP and fli1:EGFP zebrafish lines at 1 day post fertilization (dpf), but reduced to background levels at 2 dpf. The nkx2.5:EGFP fish line was least susceptible to Csm mediated EGFP knockdown. We conclude that at the present stage, Csm mediated knockdown is already efficient for maternal transcripts, and may compare favorably with morpholinos for such targets in zebrafish.