By Mark Osborn, Ph.D., Minnesota Stem Cell Institute, University of Minnesota
CRISPR/Cas9 is a vital part of our research at the University of Minnesota and the Cas9 recombinant protein, used at high concentration, has allowed for highly efficient modification of T-cells.
By introducing a Cas9 nuclease guide RNA complex (RNP), we target a specific spot in the genome, where the nuclease cuts the DNA. The DNA break is repaired in one of two ways: homologous recombination, which is high-fidelity, or non-homologous endjoining (NHEJ), which is more error-prone.
When DNA breaks occur, it’s similar to someone ripping up railroad tracks, and the cell needs to glue the ragged break back together. To process the overhangs, the cell excises bases of the DNA and glues the ends together.
In this process, some DNA sequence can be lost in such a manner that a gene can be permanently inactivated. This approach is highly relevant for T-cells, an immune system cell that is educated to distinguish self from non-self.
Therefore, we rely on the T-cell’s education system to recognize and ‘ignore’ our own tissue, but if a foreign body such as bacteria comes into our body, those cells work to eradicate it.
Disruption and direction
Working in the Division of Blood and Marrow Transplantation at the University of Minnesota, a mainstay procedure is to take out cells from one person and put them in someone else. Donor T-cells transfused into a patient with leukemia will recognize the leukemic cells as foreign and target them for destruction. But since those cells come from a different person, that recognition can tip toward recognizing healthy tissue as foreign and start to destroy it.
By disrupting the T-cell receptor (TCR) using the Cas9 protein and a TCR gRNA, we can prevent T-cells from reacting in a negative fashion. We then augment their tumor eradication ability with a second molecule called a chimeric antigen receptor.
While this example for TCRs has therapeutic implications, it also has basic biological implications: the ability to knock out candidate proteins allows us, from a discovery standpoint, to assess their function. As such, knocking out a candidate gene and subsequent loss of protein allows us to study what the effects are on the cell.
Higher concentrations, fewer cells
When we deliver the RNP complexes to cells our preferred method is to use electroporation. We expose the cells to an electrical current, which makes them temporarily porous, and essentially shoot the material into the cell. When the Cas9 protein was first available, it was available at a tenth of the concentration of what we now use, and had a lower rate of gene repair or knockout efficiency.
The entire process of mixing the cells and the proteins is affected by volume. The smaller the volume, the better the effect we can get. If we need to add more protein because of its low concentration, it dilutes the mixture and the transfer is less efficient. If we can add the same amount or less volume of the Cas9 because of its high concentration, our rates of gene modification increase helping us accomplish more in our research.
If the higher concentration Cas9 protein was unavailable, we would have to scale up our processes, which would require more cells and other materials, as well as time and money. Smaller numbers of cells with more protein is the ideal approach, which is what the high concentration Cas9 allows us to do.