Kampmann, Martin (University of California, San Francisco (UCSF))
Ludwig, Connor (University of California, San Francisco (UCSF))
Tian, Rulin (University of California, San Francisco (UCSF))
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.
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.
This plasmid could be used to screen for drug targets in human iPSC derived disease cellular models.