Knock-out of a gene of interest is a useful gene-editing strategy to validate a gene function, to develop a synthetic lethality model or directly mimic a cancer cell in drug screening. Unfortunately, non-dividing cells and hard-to-transfect cells are a major challenge for such genome-editing. However, there is an ideal solution that deserves to be more popular, the use of adenovirus.

This month, a new leap forward in gene editing has been published in Nature under the title ‘Search-and-replace genome editing without double-strand breaks or donor DNA’. DOI: 10.1038/s41586-019-1711-4. Let’s take a look at prime editing to get a clear overview.

Gene knock-out, which is gene editing leading to loss of function, just requires delivering into cells the 2 CRISPR system actors: the CAS9 endonuclease and the specific guide RNA to target the gene of interest. Thus, simple transfection of CleanCap CAS9 mRNA and gRNA followed by cell isolation with the smart aliquotor is a convenient and robust method. Howevery, what can you do if the cells are hard-to-transfect?

ORF expression in Mammalian cells is very useful for many applications: protein production with HEK293, over-expression of tagged protein for immuno-tracking or immuno-precipitation, development of a cell line for drug discovery such as a cancer model expressing PD-1 or PD-L1…

Unfortunately, plasmid delivery into primary cells and cell lines is often challenging, providing low or even no transfection efficiency. The solution is a robust lentiviral system.

So, how do you identify the right lentiviral system? With the titer! The higher is the better. 10⁷TU/ml is ok… and 10⁸TU/ml is very good.