A new publication in the Journal of Experimental Medicine cites using cryopreserved NK cells sourced from Cellero in a study focused on improving genome editing techniques.

The study’s authors, who hail from National Taiwan University, work on cancer immunotherapy. Their goal is to see the full potential of NK cells realized.

Adoptive immunotherapy is proving to be a powerful weapon in the fight against cancer. CAR T cell therapy is perhaps one of the most successful and well-known examples of this strategy. T cells isolated from cancer patients are modified using lentiviral vectors. These vectors are used to introduce modified genetic sequences into T cells via transfection. The genetically modified T cells can recognize and attack cancer cells more easily than unmodified cells.

NK cells, like T cells, can recognize and destroy cancer cells. Unlike T cells, they do not rely on HLA matching to function, an advantage that makes immune cell donation from donors to patients safe from the serious complications of graft-versus-host disease. The drawback to using NK cells, however, is that NK cells are difficult to genetically engineer. The research group in Taiwan hopes to change that by taking advantage of new advances in CRISPR technology.

CRISPR-Cas9 is currently the most widely used tool for gene editing. It gives scientists a relatively straightforward way to “edit” a section of DNA in the target cell’s genome. The cell will then translate the modified gene into a functional protein that can recognize and bind to cancer cells. Unfortunately, the lentiviral vector-CRISPR strategy does not work well with NK cells. Uptake of lentiviral vectors is unpredictable in these cells at best; and NK cells are resistant and highly sensitive to incorporating DNA donated from anyone other than patient from whom they were collected.

The research team therefore decided to design a modified CRISPR platform that uses nucleofection rather than traditional transfection. Nucleofection is an electroporation-based transfection method which enables transfer of nucleic acids such as DNA and RNA into cells by applying a specific voltage and reagents. To carry out their studies, the scientists acquired cryopreserved NK cells from Cellero. They consciously made the choice to use cryopreserved cells due to their interest in developing an off-the-shelf NK cell product with simplified storage and transport logistics. The researchers stress that proper thawing technique is critical to maintaining optimal cryopreserved NK cell viability.

A feeder-cell free expansion protocol was developed so that subsequent cell subtype population and gene editing analysis would be simplified. Expansion rates, cell surface markers, and in-vitro cytotoxicity were tracked for cells collected from 5 different donors. The team then set out to systematically optimize gene editing conditions for human NK cells.

Confirming the results of previous studies, combining CRISPR-Cas9 technology with conventional plasmid-based transfection simply did not work for NK cells. Similarly, combining the gene-editing technology with lentiviral vector delivery resulted in variable, inefficient genetic modification. Combining nucleofection with CRISPR-Cas9 technology, on the other hand, showed much more promising results. The researchers were able to achieve efficient gene knockout in NK cells while preserving high cell viability. They were also able to show robust gene knock-in of an HA affinity tag and a gene for a green-fluorescent reporter protein.

This work represents the first known example of a successful NK cell expansion and CRISPR genetic modification platform. The fact that the research group were able to accomplish this using cryopreserved NK cells as starting material highlights the potential for the technique to be widely adopted for cell therapy applications.

Cellero is pleased to have supported this work by providing high quality cryopreserved immune cells. Please visit our website to learn more about our wide selection of immune cell products.



  1. Huang R., et al. A robust platform for expansion and genome editing of primary human natural killer cells. Journal of Experimental Medicine. 218(3); 1-20. 2021.

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