What is the best transfection method for RNAi or CRISPR/Cas9 on primary keratinocytes? What is the maximum efficiency achieved?
This kit (https://altogen.com/product/keratinocyte-transfection-reagent-keratinocytes/) is designed for Keratinocytes and includes a CRISPR/Cas9 information sheet and transfection protocol. Usually primary cells are difficult to transfect, and CRISPR/Cas9 generally has less than 15% efficiency (because it has to enter the nucleus). Maybe try adding an NLS sequence to your Cas9 so that it can enter the nucleus more efficiently.
We have succesfully used Neon for these experiments. We have obtained transfection efficiencies up to 70%. http://www.thermofisher.com/fi/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system.html
Thanks. The Thermo Fisher rep is coming to demo the Neon system soon.
Lonza has a ready-to-use protocol for efficient gene transfer into neo and adult keratinocytes with 70% transfection efficiency https://www.lonza.com/products-services/bio-research/transfection/nucleofector-kits-for-primary-cells/nucleofector-kits-for-primary-dermal-cells/nucleofector-kits-for-human-keratinocytes-nhek.aspx The same protocol does work well for genome editing and CRISR/Cas9 complexes.
This kit (https://altogen.com/product/keratinocyte-transfection-reagent-keratinocytes/) is designed for Keratinocytes and includes a CRISPR/Cas9 information sheet and transfection protocol. Usually primary cells are difficult to transfect, and CRISPR/Cas9 generally has less than 15% efficiency (because it has to enter the nucleus). Maybe try adding an NLS sequence to your Cas9 so that it can enter the nucleus more efficiently.
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