Fig. 2 High Efficiency Transfection with a standard CRISPR/Cas9 EF1a-GFP DNA construct. NCRM-1iPS cells transfected using EditPro Stem with a ~10.5 kb plasmid expressing Cas9 and GFP.
Fig. 3 Transfection with EditPro? Stem Transfection Reagent Results in Effective Editing. Cells were transfected with Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay.
To allow flexibility in choice of pluripotent stem cell culture system, such as plating on other matrices or in other commercial media, we optimized transfection in suspension using a "reverse transfection" technique, for maximum transfection of all the cells in culture. Adherent cells grown in colonies on these matrices do not evenly transfect due to limited access of the reagent to the inner cells of the colony. Figure 3 illustrates how co-transfection of modified GFP mRNA and Cas9 RNP using the EditPro Stem reagent consistently produces >90% transfection efficiency as monitored by GFP in stem cells plated on vitronectin in PluriQ? G9? Maintenance Medium and on Geltrex in mTeSR?1.
Fig. 4 Transfection in Suspension: Expression of eGFP mRNA co-delivered with Cas9 Protein, tracrRNA and crRNAs Emx1 in human ESCs plated on A. vitronectin in PluriQ G9 Maintenance Medium or B. Geltrex in mTeSR
Using the transfection in suspension protocol, the modified Cas9 mRNA, tracrRNA and a modified GFP mRNA were co-transfected. The cells were then analyzed by T7Endo1 assay to look at the effectiveness of editing.
Fig. 5 Cas9 mRNA Transfection into iPSC Cells with EditPro? Stem: 200,000 cells were transfected in suspension using EditPro? Stem with 250 ng Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay.
Fig. 6 Cas9 Protein (RNP) Transfection of human iPS and ES cells with EditPro? Stem: 200,000 cells were transfected in suspension using EditPro? Stem with Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). Genome-modification was analyzed using the T7Endo 1 assay
EditPro? Stem provides superior delivery into neural stem cells. Using iPS-derived human neural stem cells and the protocol for transfection in suspension with subsequent plating on Geltrex in NeuralX? NSC Medium Supplemented with GS22? we see >90% efficiency by GFP reporter and excellent editing using either Cas9 mRNA or Cas9 Protein (RNP) as evaluated by T7Endo 1 assay.
Fig. 7 Gene Editing by co-Transfection using EditPro? Stem Transfection Reagent in Neural Stem Cells: Neural Stem cells were transfected in suspension using EditPro? Stem with 250 ng Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). GFP was observed after 24 hours. Genome-modification was analyzed using the T7Endo 1 assay.
Features:
High Efficiency – delivering DNA, RNA and/or protein
Low toxicity and does not affect pluripotency
Easy to use – no special equipment required
Cost Effective – effective at low amounts of reagent resulting in low cost/well
Kehler, J., Greco, M., Martino, V., Pachiappan, M., Yokoe, H., Chen, A., Yang, M., Jessee, J., Gotte, M., Milanesi, L., Albertini, A., Bellipanni, G., Zucchi, I., Reinbold, R. A. and Giordano, A. (2016), RNA-Generated and Gene-Edited Induced Pluripotent Stem Cells for Disease Modeling and Therapy. J. Cell. Physiol. 2016 Sep 15. doi: 10.1002/jcp.25597. [Epub ahead of print]
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