How to transfect hiPSCs with gRNA and Cas9 protein


#1

At Allen Institute, our general guidelines for CRISPR RNP Transfection include:

  • Good hiPSC morphology to start

  • For gene-editing we find the highest efficiency by pre-complexing crRNA and tracrRNA in advance of transfection

  • Recommend ~75% confluence for hiPSCs using the Neon Transfection System

  • Pre-treat cells with mTESR1 and Rock-inhibitor a 1-6 hours before transfection

  • Recommend a 1:1 molar ratio of the crRNA:tracrRNA duplex with the Cas9 protein

For more info, download our SOP: RNP transfection v1.0.pdf


#2

Also, refer to this article by the scientists at the Allen Institute for Cell Science


#3

For more detailed methods information, you can also refer to this JoVE article by the Allen Institute for Cell Science team