How to transfect hiPSCs with gRNA and Cas9 protein

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

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

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

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