By Krishanu Saha, Ph.D., Department of Biomedical Engineering & Wisconsin Institute for Discovery, University of Wisconsin-Madison
At the University of Wisconsin - Madison, one focus of our group is understanding and optimizing CRISPR-Cas9 gene editing for therapeutic and disease modeling applications. To conduct our research, we need reliable, consistent and highly efficient Cas9 protein.
Model system for Cas9 gene editing
To perform targeted gene editing, the Cas9 protein, which cuts the genome, and a guide RNA (gRNA) that encodes where in the genome to cut, need to be complexed together into a ribonuclear protein (RNP) complex and transfected to cells to reach the nucleus. Once the DNA is cut, imprecise DNA repair may cause disruption at the cut site, which can result in knock out of a gene.
Knocking out genes helps biologists understand their function within human cells. Further, in therapeutic application, knock out of a pathogenic gene may treat an inherited disorder, which is the case for sickle cell anemia and muscular dystrophy. However, for many of these cases, high efficiency editing is required to get the required effect.
One way in which we can easily test gene editing efficiency is by using human reporter cells, whose nuclei glow red. These cells have been engineered to express the gene mCherry in their nuclei, making the nuclei appear red when viewed under a fluorescence microscope.
By making a gRNA that targets the mCherry gene for disruption and transfecting it with Cas9, successful cutting can be measured by the percentage of cells losing their mCherry fluorescence, as illustrated in Figure 1 (use the magnifying glass icon to show a larger version).
High-efficiency Cas9 for gene editing
To optimize different aspects of the gene editing process, we needed to make sure that the Cas9 protein we use is efficient and generates reproducible results. We screened multiple variants of Cas9 protein from different companies to find one we can reliably use for our research. We used our model system in human embryonic kidney (HEK) cells, using lipofection, to compare the efficiency of mCherry knockout.
Figure 2 shows some of the mCherry knockout efficiencies we measured using flow cytometry. We saw high knockout efficiency of the engineered Cas9 variants from Aldevron, compared with the competitor’s product. The nuclear localization signal (NLS) on Cas9 helped translocation of the Cas9 RNP to the nucleus and increase efficiency.
Furthermore, the high Cas9 concentration of Aldevron’s formulation allows us to effectively use the required amounts of protein without diluting it extensively with our other reagents or cell media. After obtaining high repeatability of results with these proteins, we are now able to quickly advance our disease modeling and regenerative medicine research.