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.

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.

Hybrid drug inducible CRISPR/Cas9 technology, HIT-Cas9

Zhao et al. revealed tight and effective drug control of the CRISPR Cas9 based on 4-Hydroxytamoxifen.

They built a fusion between a mutated human estrogen receptor (ERT2) and the Cas9 endonuclease, which makes the CRISPR Cas9 dependent on 4-hydroxytamoxifen.