Type III CRISPR-Cas System for Robust RNA Knockdown and Imaging in Eukaryotes

Type III CRISPR-Cas systems recognize and degrade RNA molecules using an RNA-guided mechanism that occurs widely in microbes for adaptive immunity against viruses. The inventors have demonstrated that this multi-protein system can be leveraged for programmable RNA knockdown of both nuclear and cytoplasmic transcripts in mammalian cells.

Using single-vector delivery of the S. thermophilus Csm complex, RNA knockdown was achieved with high efficiency (90-99%) and minimal off-targets, outperforming existing technologies of shRNA- and Cas13-mediated knockdown. Furthermore, unlike Cas13, Csm is devoid of trans-cleavage activity and thus does not induce non-specific transcriptome-wide degradation and cytotoxicity. Catalytically inactivated Csm can also be used for programmable RNA-binding, which the inventors exploit for live-cell RNA imaging. This work demonstrates the feasibility and efficacy of multi-subunit CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes.

Website

https://techtransfer.universityofcalifornia.edu/NCD/32820.html?utm_source=AUTMGTP&utm_medium=webpage&utm_term=ncdid_32820&utm_campaign=TechWebsites

Advantages

The advantages include:

  • yields robust, highly efficient RNA knockdown with minimal off-targets and no cell toxicity
  • cheap, easy to use, and easy to deliver as an all-in-one vector
  • allows for gene perturbation at the transcript (RNA) level
  • efficiently knocks down both cytoplasmic and nuclear RNAs

Potential Applications

This invention has the potential to completely supplant RNAi and Cas13-based RNA knockdown technologies.

Suggested uses include:

  • research purposes requiring gene perturbation at the transcript (RNA) but not genome (DNA) level.
  • efficiently knocking down both cytoplasmic and nuclear RNAs.
  • RNA imaging in live cells.
  • RNA detection for diagnostic purposes.
  • utilizing the enzyme’s ssDNase and/or cyclic oligoadenylate synthesis activities.

Contact Information

Name: Terri Sale

Email: terri.sale@berkeley.edu

Phone: 510-643-4219