Description of the molecular mechanisms underlying CRISPRi. [Cell Stem Cell, doi:10.1016/j.stem.2016.01.022]
Description of the molecular mechanisms underlying CRISPRi. [Cell Stem Cell, doi:10.1016/j.stem.2016.01.022]

Combining powerful technologies in biology can often have an exceptionally synergistic effect that leads to fundamental breakthroughs in how researchers look at normal metabolic pathways or aberrant disease states. This is what researchers from the Gladstone Institute hope will emerge from their recent use of a relatively new variation of the CRISPR-Cas9 system on the genome of induced pluripotent stem cells (iPSCs)—offering a significant technological advance in creating cell models of genetic diseases.    

In the newly published study, researchers used a modified version of CRISPR called CRISPR interference (CRISPRi) to inactivate genes in iPSCs and heart cells created from iPSCs. The method, first reported in 2013 by Stanley Qi, Ph.D., who is a co-author on the current paper, significantly improves the original CRISPR-Cas9 system by allowing genes to be silenced more precisely and efficiently. Additionally, CRISPRi also offers the flexibility of reversing and carefully controlling the amount of gene suppression.

The standard CRISPR system uses the Cas9 protein to delete a precise part of the genome through small cuts in a cell's DNA. CRISPRi builds on this technology by using a special deactivated version of the Cas9 protein along with an additional inhibitor protein, Krüppel-associated box, or KRAB. Together, these proteins sit at the target spot on the genome and suppress gene expression without excising the DNA.

“We developed clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs),” the authors wrote. “CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles.”

Surprisingly the investigators found that temporarily silencing gene expression in this manner was much more consistent than permanently cleaving the genome.

“We were amazed by the dramatic difference in performance between the two systems,” remarked senior study author Bruce Conklin, M.D., senior investigator at the Gladstone Institute of Cardiovascular Disease and Roddenberry Stem Cell Center. “We thought that permanently cutting the genome would be the more effective way to silence a gene, but in fact, CRISPRi is so precise and binds so tightly to the genome that it is actually a better way to silence a gene.”

In the current study, the Gladstone scientists compared how well CRISPRi and CRISPR-Cas9 silenced a particular gene that controls iPSC pluripotency. They discovered that CRISPRi was much more efficient than CRISPR-Cas9, because, in 95% of the cells created using CRISPRi, the target gene was silenced, compared to only 60–70% of cells grown from traditional CRISPR-Cas9 editing. Moreover, CRISPRi also did not cause any off-target changes in gene expression, like undesired insertions or deletions to the cell's genome, which is typically a cause for concern with CRISPR-Cas9.

The findings from this study were published recently in Cell Stem Cell in an article entitled “CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs.”

Another advantage of CRISPRi is that it can act as a toggle switch, enabling the scientists to reverse gene suppression by simply removing the chemical that turns on the gene inhibitor. In the current study, the researchers were able to tune how much they silenced a gene by changing the amount of the chemicals they added. Both of these results support much more versatile investigations into the role of individual genes that affect development and disease.

“CRISPRi holds a major advantage in making disease-relevant cell types,” explained lead author Mohammad Mandegar, D.Phil., research scientist at Gladstone. “Using this technology, we can mimic disease in a homogeneous population of heart cells created from iPSCs. This development allows us to study genetic diseases more easily and potentially identify new therapeutic targets.”

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