Novel CRISPR system modifies how stem cell genome is read

Scientists from the Gladstone Institutes (CA, USA) have utilized a variation of the CRISPR-Cas9 system to modify how the genome of induced pluripotent stem cells is expressed.

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Mar 11, 2016
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Researchers from Gladstone Institutes (CA, USA) have combined two of the most powerful biological tools of the last hundred years, altering for the first time how the genome of induced pluripotent stem cells (iPSCs) is read, utilizing a variation of the CRISPR-Cas9 system. The study offers a major advance in creating cell models of genetic diseases.

The team employed a modified version of CRISPR called CRISPR interference (CRISPRi) to inactivate genes in both iPSCs and heart cells developed from iPSCs. The method was first reported in 2013 by Stanley Qi, co-author of the current study, and it significantly improves the original CRISPR-Cas9 system by allowing the silencing of genes with increased precision and efficiency. CRISPRi is also more flexible, as it allows the user the reverse and carefully control the amount of gene suppression.

CRISPRi builds on the standard CRISPR system by using a novel deactivated version of the Cas9 protein and an addition inhibitor protein, KRAB. These proteins sit at the target point on the genome and suppress gene expression without cutting the DNA, a system which surprisingly was much more consistent than permanently cleaving the genome.

"We were amazed by the dramatic difference in performance between the two systems," described 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."

The researchers compared how well CRISPRi and CRISPR-Cas9 silenced a particular gene, responsible for controlling the capability of iPSCs to differentiate into multiple cells types. In more than 95% of the cells created using CRISPRi, the target gene was silenced, in comparison with only 60–70% of cells grown from standard CRISPR. The novel technique also did not cause off-target changes in gene expression, such as deletions or insertions to the genome, which is a shortcoming of CRISPR-Cas9.

The team was also able to demonstrate that CRISPRi acted as a ‘switch’, enabling scientists to reverse suppression of genes by removing the chemical that activates the gene inhibitor. The researchers were able to ‘fine-tune’ the degree to which genes were silenced, by carefully modifying the amount of chemicals added to the system. These results support more versatile research into the role of specific genes in development and disease.

"CRISPRi holds a major advantage in making disease-relevant cell types," explained first author Mohammad Mandegar. "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."

Sources:

Mandegar MA, Huebsch N, Frolov EB et al. CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs. Cell Stem Cell doi:10.1016/j.stem.2016.01.022 (2016) (Epub ahead of print); https://gladstone.org/about-us/news/modified-form-crispr-acts-toggle-switch-control-gene-expression-stem-cells
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Stella Bennett

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