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.
Are you looking for a simple means to boost the efficiency of gene editing and also to reduce the off-target? Yes, it does exist – in one word: 4-hydroxy Tamoxifen. That’s the HIT-CAS9.
They built a fusion between a mutated human estrogen receptor (ERT2) and the Cas9 endonuclease, which makes the CRISPR Cas9 dependent on 4-hydroxytamoxifen.