Targeted RNA Knockdown by a Type III CRISPR-Cas Complex in Zebrafish

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 endoribonuclease can be used for programmable mRNA transcript degradation in eukaryotes. Using zebrafish as the animal model and Csm complex as the CRISPR endoribonuclease, we targeted 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 also tested Csm mediated knockdown on the endogenous tdgf1 (oep) transcript. At optimal Csm dose, we observed a penetrance of the characteristic one-eyed phenotype at greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown with no significant off-target effects in our model. Overall design: Zebrafish embryos from the transgenic line Tg(ddx4:ddx4-EGFP) on the background of ABTL were injected with the Csm complex targeted against the EGFP transcript or mock solution (StCsm buffer without the proteins). The RNA was isolated from the pooled embryos at three time points: 128 cell stage, 5 hpf and 24 hpf. Three replicates were obtained from each experimental condition and time point. Pair-end sequencing was performed on the Illumina NextSeq platform.