A CRISPR/Cas9-based system for reprogramming cell lineage specification.
Bottom Line: Gene activation by the CRISPR/Cas9 system has the potential to enable new approaches to science and medicine, but the technology must be enhanced to robustly control cell behavior.Targeted activation of the endogenous Myod1 gene locus with this system led to stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes.The levels of myogenic marker expression obtained by the activation of endogenous Myod1 gene were comparable to that achieved by overexpression of lentivirally delivered MYOD1 transcription factor.
Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA.Show MeSH
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Mentions: To test whether transient expression of VP64dCas9-BFPVP64 is sufficient to induce sustained expression of Myod1, we induced transgene expression between days 2 and 10 posttransduction. We detected an initial robust upregulation of VP64dCas9-BFPVP64 expression followed by a gradual downregulation suggesting transgene silencing in the reprogramming cells (Figure 3A). Doxycycline removal coincided with rapid VP64dCas9-BFPVP64 decline to levels measured prior to initial induction. VP64dCas9-BFPVP64 expression also coincided with an increase in Myod1 expression. Importantly, Myod1 mRNA levels remained elevated for the duration of the 18-day experiment even after doxycycline removal (day 10), suggesting that maintenance of endogenous activation of Myod1 is stable and independent of VP64dCas9-BFPVP64 activity.
Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA; Department of Biomedical Engineering, Columbia University, New York, NY 10027, USA.