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.

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

“…Further studies would be needed to clarify the mechanism of NgAgo's RNA interference and the interaction between NgAgo–gDNA complex and the RNA target. In addition, other similar RNA‐guided riboendonucleases, such as Cas13a/C2c2 and Csm, were recently reported for RNA silencing application. Those enzymes including NgAgo may serve as potential alternatives to RNAi, but more studies, especially more in vivo characterization, would be needed.…”

Harnessing “A Billion Years of Experimentation”: The Ongoing Exploration and Exploitation of CRISPR–Cas Immune Systems

“…85 And although the multisubunit nature of Csm/Cmr complexes has thus far precluded biotechnological applications in eukaryotic cells, a recent preprint describes the use of recombinant Csm complexes to achieve targeted RNA knockdown in zebrafish embryos. 236 Cas13, which naturally targets RNA within type VI systems, is proving to be a far more powerful enzyme for RNAspecific applications (Fig. 3C).…”