In March 2016, Mark J Osborn et al published in Molecular Therapy a major article for genome editing (doi:10.1038/mt.2015.197), about knock-out of CD3 in human T-cells. The goal is to improve T-cell-based immunotherapies to fight tumours using engineered allogenic T-cells from healthy donors. It is a very good example of how CRISPR-CAS9 can help medecine. And even if you are not very comfortable with CAR T-cells and the treatments of malignancies, I would recommend you read it and especially take a look at figure 2. Indeed, dear friends of genome editing, the authors made a clear and fair comparison of several KO strategies, covering all the main options. Thus, it is not only a major step for anti-tumour treatments but it is also an excellent overview that reveals the best approaches. So, before reading this post any further, you might like to read the article mentioned above.

Combo of modified CAS9 mRNA and synthetic gRNA

Now, if you have read the article by Mark J Osborn et al, you won’t be surprised that I definitely recommend you use a combo of CAS9 mRNA with synthetic and 2’OMe modified gRNA for the best KO.

RNP CRISPR-CAS9 complexe

2’Me/PS modified gRNA in complexe with CAS9

The red stars indicate 2’OMe and Phosphorothioate modifications on the complete gRNA (~100nt). tebu-bio is the unique commercial source in Europe able to provide it. Competitors are not able to make such long gRNA with the same quality.

Furthermore, we know now that to deliver the CAS9 protein directly is not an efficient approach. Delivery of CAS9 mRNA is far better. Nevertheless, producing mRNA can be a challenge, but no need to worry, there is an optimised CAS9 mRNA ready-to-use.

Such a combo can be used for cytoplasmic injection into Mouse oocytes. For the transfection of cells,we can help with the delivery using our dedicated reagent, mRNA-in. A neuronal cell type version  and a stem cell version  have been developed too.

Performances of transfection over three cell types

Transfection of eGFP mRNA with mRNA-in

All-in-one vector expressing CAS9 and gRNA

In the perspective of therapies, it is highly preferable to avoid vectors to deliver the CRISPR-CAS9 system to ensure genome integrity. But for all other projects, why not? Especially when we know it is a highly efficient way.

We offer an all-in-one vector expressing CAS9 and the specific gRNA with a convenient mCherry reporter to monitor the transfection efficiency.

pCRISPR-CG01

pCRISPR-CG01, all-in-one vecteur expressing CAS9 and gRNA

It’s a budget-friendly approach starting from 330 euros.

For the transfection, there is also a dedicated solution called DNA-in CRISPR.

In conclusion, you can find all the reagents you need for the 2 best methods to obtain a knock-out!

Furthermore, I’ll be more than happy to offer assistance if needed, please feel free to leave your questions and comments below!

Dimitri Szymczak, PhD
Written by Dimitri Szymczak, PhD
Dimitri Szymczak is a Technical Support Specialist and Product Manager at tebu-bio, and a fan of capoeira in his spare time.