Technology ID
TAB-3710
Development of a plasmid (pRT029) to enable CRISPRi gene knowndown in human stem cells (iPSCs)
E-Numbers
E-207-2018-0
Lead Inventor
Ward, Michael (NINDS)
Co-Inventors
Kampmann, Martin (University of California, San Francisco (UCSF))
Ludwig, Connor (University of California, San Francisco (UCSF))
Tian, Rulin (University of California, San Francisco (UCSF))
Applications
Therapeutics
Research Materials
Diagnostics
Lead IC
NINDS
ICs
NINDS
The invention relates to a plasmid that enables gene knowndown (via CRISPRi) in human induced pluripotent stem cells (iPSCs), including derived cell types such as neurons. The plasmid contains homology arms to direct insertion of a cassette into the CLYBL safe-harbor locus in the human genome. The cassette expresses CRISPRi machinery using a CAG promoter. The CRISPRi machinery consists of double degron-tagged dCas9-BFP-KRAB. Addition of the small molecule trimethoprim to cell culture media stabilizes the degron and thereby increases dCas9-BFPKRAB levels, enabling CRISPRi activity.
Commercial Applications
No tools currently exist to enable inducible, targeted gene silencing via CRISPR-related technology in iPSC-derived cells. The plasmid described here encodes an enzymatically-dead variant of Cas9, fused to a degradation domain that can be stabilized by the addition of trimethoprim, a small molecule.
Competitive Advantages
This plasmid could be used to screen for drug targets in human iPSC derived disease cellular models.
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