Why is a clonally pure cell line so important in CRISPR research?
Never far from scientific and mainstream news, CRISPR is regularly hailed as one of the most important scientific discoveries of our time. With potential applications across wide-ranging fields from medicine to agriculture, being able to control gene expression could provide treatments for a number of genetic diseases and solve global food shortages in times of continuing environmental change. Most recently, the ethics of this research have once again hit the headlines as a Chinese researcher claims to have created the first genetically edited babies. Whilst discussion continues over ethical implications, further research to support and inform this is ever more important.
CRISPR/Cas9 consists of two components: a Cas protein (often Cas9) and a guide RNA (gRNA). After binding of the gRNA to the targeted DNA sequence, the Cas protein generates a double-stranded break at the targeted site. At this point, researchers can knock-out expression of the target gene or knock-in an alteration to this DNA. This process is error-prone, often resulting in cells in which the gene is either incompletely knocked out or is untransfected, and which carry unwanted background mutations. It is therefore essential to be able to isolate a single cell clone and sequence verify the clone.
Traditional methods of cell line development include limiting dilution and flow cytometry, but both have their drawbacks. They are not only time consuming but typically exhibit lower efficiencies for isolating viable single cells or poor resultant outgrowth, and they rely on either probabilities or indirect measurements, such as whole well imaging, to confirm clonality. The clonality of the resulting cell line must be proven in order for these cell lines to be reliably used for disease models and for target validation studies. Some of the most interesting cell types being studied for cell engineering in drug discovery and cell therapy are also some of the most challenging to isolate as single cells and then to successfully grow out, for example, iPSC cells.
Solentim’s VIPS™ is a unique all-in-one instrument which seeds individual cells into 96 well plates, dispensing nano-litre droplets at a dispensing pressure of less than 1 psi, resulting in high cell survival rates and a high seeding efficiency (over 80%, cell-type dependent). The gentle mode of dispensing is also important so as not to disturb the cell phenotype. Not only does the instrument improve efficiency and save researchers time and money, it also verifies the clonality of the resulting cell lines. This is achieved by imaging the cell droplets after they are dispensed at the bottom of a dry well. These cell droplet and whole well images are included in the Clonality Report. This documentation of the clonality will be an important part of drug companies’ internal record-keeping for these cell lines and to support future potential IP.
The science and research behind CRISPR progresses every day; it is vital to develop equipment to support and facilitate these advances. The ability to easily isolate and verify a single cell containing a specific edit significantly aids researchers, making subsequent downstream experiments on these cell lines much more reliable and predictive.