Q:

Which program should I use for co-transfection of RNP and CRISPR/Cas9 complexes ? The paper "Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells from Aikiko Seki and Sascha Rutz showed different programs as you have in the Lonza optimized Nucleofector Protocols?

A:

Their publication in the Journal of Experimental Medicine is an advanced optimization for gene knock out in primary human and mouse T cells. The final protocol achieved 85-98% gene knock out for several tested receptors.

The group has tested and optimized many parameters, which are important for successful genome editing. Not only the selection of RNPs and CRISPR complexes, but also cell culture conditions and many different transfection conditions. Finally, they also titrated the best ratio of the different components for genetic modification of the cells.

For unstimulated mouse T cells they have finally used DS-137 in combination with P4 Primary Cell 4D-Nucleofector™ Solution. For human unstimulated, resting T cells they identified EH-100 in combination with P2 Primary Cell 4D-Nucleofector™ Solution.

The ready-to-use protocols are cell type specific and not substrate specific. That means you can use the same program for Nucleofection™ of small and large DNA vector, DNA fragments, sgRNA, siRNA, peptides and proteins. Basically, we recommend using the ready-to-use protocol for the specific cell type. In this paper they did many changes in the protocol, used 1-10 million cells in the small scale set up and increased the transfection volume from 20 to 35ul. These are changes which could impact the transfection parameters, so for exactly this set up it might be a good choice. We recommend testing the optimized protocol and probably those parameters with our control pmax-GFP vector to see if there are major differences

Categories:
Transfection
Research Areas:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Basic Research