Generation of Inducible CRISPRi and CRISPRa Human StromalStem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation

الملخص EN

Background.

Human bone marrow stromal/stem cells (hMSCs, also known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes.

However, mechanistic studies employing hMSC have been hampered by the difficulty of deriving genetically modified cell lines due to the low and unstable transfection efficiency.

Methods.

We infected hMSC with a CRISPR/Cas9 lentivirus system, with specific inducible dCas9-coupled transcription activator or repressor: dCas9-KRAB or dCas9-VP64, respectively, and established two hMSC lines (hMSC-CRISPRi and hMSC-CRISPRa) that can inhibit or activate gene expression, respectively.

The two cell lines showed similar cell morphology, cell growth kinetics, and similar lineage differentiation potentials as the parental hMSC line.

The expression of KRAB-dCas9 or VP64-dCas9 was controlled by the presence or absence of doxycycline (Dox) in the cell culturing medium.

To demonstrate the functionality of the dCas9-effector hMSC system, we tested controlled expression of alkaline phosphatase (ALP) gene through transfection with the same single ALP sgRNA.

Results.

In the presence of Dox, the expression of ALP showed 60-90% inhibition in hMSC-CRISPRi while ALP showed more than 20-fold increased expression in hMSC-CRISPRa.

As expected, the ALP was functionally active and the cells showed evidence for inhibition or enhancement of in vitro osteoblast differentiation, respectively.

Conclusion.

hMSC-CRISPRi and hMSC-CRISPRa are useful resources to study genes and genetic pathways regulating lineage-specific differentiation of hMSC.