The CRISPR-Cas9 system is one of the most promising biological tools that we have. It allows scientists to investigate and manipulate genes inside cells and do so with unprecedented precision—it has thrust us into a new age of genetic engineering. Now, scientists have modified it further, allowing it to be used to modify the genomes of induced pluripotent stem cells (iPSCs).

In a study published in Cell Stem Cell, researchers explain how they used a modified version of CRISPR, called CRISPR interference (CRISPRi), to inactivate genes in iPSCs and heart cells created from iPSCs. This development would allow researchers to precisely and efficiently turn off genes using CRISPRi.  Furthermore, it gives researchers control over the amount of gene suppression, and allows them to reverse it if needed.

How it Works

The CRISPR system uses the protein Cas9 to delete a precise part of the genome by making small cuts in a cell's DNA. CRISPRi goes beyond this by using a special deactivated version of the Cas9 protein along with an inhibitor protein, KRAB. The two proteins then sit at the target spot within the genome and suppress gene expression without any cuts. The end result is a more consistent gene suppression compared to cutting it outright.

"We were amazed by the dramatic difference in performance between the two systems," said senior author Bruce Conklin. "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."


A colony of iPSCs. Red and green are markers of pluripotency. Credit: Salk Institute for Biological Studies

The researchers found that CRISPRi was more efficient with more than 95% of cells created using CRISPRi showing gene suppression. In contrast, CRISPR-Cas9 resulted only in target gene suppression in around 60%-70% of cells.

CRISPRi also did not cause any off-target changes, such as insertions or deletions in the cell’s genome, a concern during use of CRISPR-Cas9. CRISPRi was also able to work in other cells aside from iPSCs, such as T cells and heart cells made from stem cells.

CRISPRi was effectively acting like a toggle switch which scientists could use to reverse the gene suppression by removing the chemical that activates the gene inhibitor. By fine-tuning the amount of chemicals added, the researchers were able to control the extent of the effects. The results could allow for more versatile investigations of the role of genes in diseases and genetic ailments.

"CRISPRi holds a major advantage in making disease-relevant cell types," said first author Mohammad. "Using this technology, we can mimic disease in a homogenous 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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