From horses in the farmyard to yeast in beer production, human beings have a long history of putting nature’s gifts to work. This can also be said of CRISPR technology, which utilizes a Cas9 DNA endonuclease and a guide RNA to target and destroy specific DNA sites.
Nature had intended a role for CRISPR in bacterial immune systems, but we soon realized its wider potential and put it to different uses. In stem cell research, one of CRISPR’s biggest successes has been the correction of faulty genes in induced pluripotent stem cells (iPSCs) before their subsequent differentiation into healthy replacement cells and/or tissues.
But therein lies a problem. Differentiation of iPSCs or embryonic stem cells (ESCs) is a tricky business, and the field has been searching for a means to gain control over differentiation-associated gene expression to promote effective and efficient cell differentiation.
In a major step in the right direction, researchers from the group of Timo Otonkoski have hacked CRISPR-Cas9 to generate designer gene activation factors that function in stem cells.
Their study, reported in Stem Cell Reports, evolved in two stages:
- Spatial control with dCas9VP192
- The group fused a Cas9 protein lacking DNA-destruction ability (dCas9) to 12 copies of a protein with potent gene activation activity (dCas9VP192)
- Adding a promoter-specific guide RNA to the mix generated a specific gene expression activator.
- Temporal Control with DDdCas9VP192
- The group next fused a destabilization domain (DD) to dCas9VP192 to allow activation of gene expression only after the addition of a stabilizing drug.
- The group then applied this system to:
- Force pluripotency gene expression in embryonic and primary cells
- Aid reprogramming of somatic cells into iPSCs through the activation of OCT4 expression
- Activate the expression of specific genes in hESCs and hiPSCs to significantly boost their differentiation potential.
This CRISPR hack has generated an effective gene expression system, which if combined with large guide RNA arrays, could effectively differentiate human stem cells into functional cell populations. The authors do note that a few tweaks to this protein hack will be required first including boosting activation efficacy, enhancing destabilization control, and reducing the protein construct size.
Read all the details on how the researchers hacked CRISPR to create this exciting new gene control method at Stem Cell Reports, September 2015.