While the DNA editing function of CRISPR/Cas9 systems may appear to hog the limelight, we believe that a new SunTag approach for CRISPR/Cas9-mediated induced pluripotent stem cell (iPSC) reprogramming via epigenome editing deserves its day in the sun!
The exciting reprogramming strategy utilizes an endonuclease dead Cas9 (dCas9) fused to a repetitive protein scaffold known as SunTag, which recruits multiple copies of effector domains fused to antibodies that recognize SunTag. Previous studies have employed this advanced strategy to edit the epigenome by promoting either DNA methylation or DNA demethylation via DNMT3A or TET1 effector domains, respectively.
Now, researchers from the lab of Sheng Ding (J. David Gladstone Institutes, San Francisco, USA) have promoted chromatin remodeling and gene expression of pluripotency-associated genes via the SunTag approach using the VP64 transcriptional activation domain or the core histone acetyltransferase (HAT) domain of p300 (p300core); a strategy known as “CRISPR activation”.
The team took to this approach to cast some light on the mechanisms controlling the reprogramming of somatic cells to iPSCs, but also in the hope of crafting a more efficient and effective reprogramming protocol. The classical methods employed to generate iPSCs exploit the forced expression of multiple transcription factors, which can obscure the sequential mechanisms at play during the acquisition of pluripotency.
Let´s delve into the highlights of this new study, which employed mouse embryonic fibroblasts (MEFs) as the target cell type:
- CRISPR activation employing dCas9-SunTag-VP64 targeted and remodeled the endogenous Oct4 and Sox2 pluripotency-associated gene loci using single guide RNAs specific for regulatory sequences
- Unexpectedly, the single act of remodeling the previously silenced Sox2 promoter triggered the expression of other pluripotency-associated genes in MEFS and permitted the establishment of the pluripotency network
- Remodeling of the Oct4 promoter and enhancer also promoted the establishment of the pluripotency network in MEFs
- Both these scenarios permitted the generation of karyotypically-normal fully pluripotent iPSC lines, as evidenced by the ability of iPSCs to contribute to chimeric animals
- Excitingly, targeting dCas9-SunTag-p300core to the endogenous Oct4 gene promoter and enhancer promoted histone acetylation and the initiation of reprogramming
- However, the authors noted a noticeable latency in transcriptional activation for the p300core domain when compared to VP64
Ding himself notes that “This is a new way to make induced pluripotent stem cells that is fundamentally different from how they’ve been created before. 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.”
He continues, “Having different options to make iPSCs will be useful when scientists encounter challenges or difficulties with one approach. 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. Now, we want to understand how this whole process spreads from a single location to the entire genome.”
Can SunTag shed some light on your iPSC research? Try switching on that table lamp and reading all the details concerning this new study over at Cell Stem Cell, February 2018.