# How to transfect hiPSCs with gRNA and Cas9 protein

**URL:** <https://forum.allencell.org/t/how-to-transfect-hipscs-with-grna-and-cas9-protein/37>\
**Category:** Gene-editing Discussion\
**Created:** [September 26, 2018, 10:49pm UTC](https://forum.allencell.org/t/how-to-transfect-hipscs-with-grna-and-cas9-protein/37 "2018-09-26T22:49:14Z")\
**Posts on this page:** 3\
**Page:** 1

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**Author:** ![cells](https://avatars.discourse-cdn.com/v4/letter/c/ecc23a/32.png) [@cells](https://forum.allencell.org/u/cells)\
**Post date:** [September 26, 2018, 10:49pm UTC](https://forum.allencell.org/t/how-to-transfect-hipscs-with-grna-and-cas9-protein/37/1 "2018-09-26T22:49:14Z")

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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](https://www.allencell.org/methods-for-cells-in-the-lab.html#sops)

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**Author:** ![kimberly.metzler](https://avatars.discourse-cdn.com/v4/letter/k/bc79bd/32.png) [@kimberly.metzler](https://forum.allencell.org/u/kimberly.metzler)\
**Post date:** [September 26, 2018, 10:51pm UTC](https://forum.allencell.org/t/how-to-transfect-hipscs-with-grna-and-cas9-protein/37/2 "2018-09-26T22:51:15Z")

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

> <https://www.ncbi.nlm.nih.gov/pubmed/28814507>
>
> We present a CRISPR/Cas9 genome-editing strategy to systematically tag endogenous proteins with fluorescent tags in human induced pluripotent stem cells (hiPSC). To date, we have generated multiple hiPSC lines with monoallelic green fluorescent protein tags labeling 10 proteins representing major cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, nuclear lamin B1, nonmuscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome-editing methodology using Cas9/crRNA ribonuclear protein and donor plasmid coelectroporation, followed by fluorescence-based enrichment of edited cells, typically resulted in \<0.1-4% homology-directed repair (HDR). Twenty-five percent of clones generated from each edited population were precisely edited. Furthermore, 92% (36/39) of expanded clonal lines displayed robust morphology, genomic stability, expression and localization of the tagged protein to the appropriate subcellular structure, pluripotency-marker expression, and multilineage differentiation. It is our conclusion that, if cell lines are confirmed to harbor an appropriate gene edit, pluripotency, differentiation potential, and genomic stability are typically maintained during the clonal line-generation process. The data described here reveal general trends that emerged from this systematic gene-tagging approach. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community.

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**Author:** ![amandah](https://avatars.discourse-cdn.com/v4/letter/a/e47774/32.png) [@amandah](https://forum.allencell.org/u/amandah)\
**Post date:** [September 27, 2018, 2:33pm UTC](https://forum.allencell.org/t/how-to-transfect-hipscs-with-grna-and-cas9-protein/37/3 "2018-09-27T14:33:49Z")

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

> **[Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using...](https://www.jove.com/video/58130/endogenous-protein-tagging-human-induced-pluripotent-stem-cells-using)**
>
> Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells...