For the first time ever, scientists from the Gladstone Institutes have used a variation of the CRISPR-Cas9 system to read the genome of induced pluripotent stem cells (iPSCs) in a different manner, which will help us advance how we create cell models of genetic diseases.
During the study, the team used a modified version of CRISPR called CRISPR interference (CRISPRi) that allowed them to inactivate genes in iPSCs and heart cells that were created from iPSCs. This ability is a significant improvement over the original CRISPR-Cas9 system, as it allows scientists to turn off genes in a more precise and effective manner, giving them the ability to control and reverse gen suppression in a more flexible manner.
While the original CRISPR system utilizes the Cas9 protein to cut cell DNA in order to delete genome regions, CRISPRi uses a unique deactivated version of the Cas9 protein combined with an inhibitor protein, KRB. When combined, these proteins can suppress gene expression by simply sitting at the target location, eliminating the need for cutting DNA. This temporary silencing is much more consistent than the permanent cleaving of the genome.
"We were amazed by the dramatic difference in performance between the two systems," said Bruce Conklin, senior author of the study. "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."
Through comparison, the team found that CRISPRi is much more efficient than CRISPR-Cas9 when it comes to silencing particular genes that control iPSC pluripotency, and it also did not cause any unwanted changes in gene expressions, a current concern with the CRISPR-Cas9 system.
The biggest advantage of CRIPSRi, however, is the ability to act as a toggle switch - when scientists want to reverse gene suppression, they simply remove that chemical that is responsible for turning the inhibitor on. In addition, altering the amount of chemicals added allows researchers to gauge how much the gene is silenced, giving them more versatility when researching the role of genetics in the development of disease.
"CRISPRi holds a major advantage in making disease-relevant cell types," concluded Mohammad Mandegar, first author of the study "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."
The findings were published in the March 10 issue of Cell Stem Cell.
