A new way of inducing pluripotency has been demonstrated by scientists based at the Gladstone Institutes. Rather than fuss with the usual transcription factors or drug-like chemicals, the Gladstone scientists, led by Sheng Ding, Ph.D., used CRISPR activation (CRISPRa). This technique, which incorporates a modified form of CRISPR gene-editing systems Cas9 enzyme, allowed Dr. Ding and colleagues to target and epigenetically remodel specific genomic locations: the Oct4 and Sox2 genes.
Remodeling either gene, the scientists discovered, turned skin cells from mice into stem cells. Detailed findings appeared January 19 in the journal Cell Stem Cell, in an article entitled “CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency.”
“…single-locus targeting of Sox2 was sufficient to remodel and activate Sox2, which was followed by the induction of other pluripotent genes and establishment of the pluripotency network,” wrote the article’s authors. “…targeted manipulation of histone acetylation at the Oct4 gene locus could also initiate reprogramming.”
CRISPRa systems incorporate a “dead” cutting enzyme and are fused to transcriptional activators. Such systems, the authors of the current study noted, were used to examine the activation of endogenous pluripotent genes in previous studies. In these studies, no induced pluripotent stem cells (iPSCs) were established.
Dr. Ding and colleagues reported that they had better results with their CRISPRa systems—the dCas9-SunTag-VP system and the dCas9-SunTag-p300core system—both of which are optimized versions of the SunTag reprogramming system. “Our results show that the epigenetic remodeling of Oct4 promoter and enhancer, either through VP64 or p300core, is sufficient to trigger reprogramming toward pluripotency,” the authors of the Cell Stem Cell article noted.
“Simultaneous remodeling of the Oct4 promoter and enhancer also triggered reprogramming,” the authors indicated. “Authentic pluripotent cell lines were established in both cases.” The authors added that their CRISPRa approach not only generates iPSCs but also sheds light on the mechanisms of cellular reprogramming. They assert that their reprogramming strategy should also work in the generation of other cell types and in other model systems, such as human cells.
“This is a new way to make iPSCs that is fundamentally different from how they've been created before,” said Dr. Ding. “At the beginning of the study, we didn't think this would work, but we wanted to at least try to answer the question: Can you reprogram a cell just by unlocking a specific location of the genome? And the answer is yes.”
When pluripotency is induced with reprogramming factors, four such factors are usually deployed. What's more, one transcription factor typically targets thousands of genomic locations in the cell and changes gene expression at each location.
“Having different options to make iPSCs will be useful when scientists encounter challenges or difficulties with one approach,” remarked Dr. Ding. “Our approach could lead to a simpler method of creating iPSCs or could be used to directly reprogram skin cells into other cell types, such as heart cells or brain cells.”
“The fact that modulating one site is sufficient is very surprising,” he said. “Now, we want to understand how this whole process spreads from a single location to the entire genome.”