Simple, fast and efficient iTOP-mediated delivery of CRISPR/Cas9 RNP in difficult-to-transduce human cells including primary T cells

•The optimized iTOP technology® is a simple, fast and efficient intracellular delivery method for CRISPR/Cas9 RNPs.•Primary human cells can be edited successfully using the iTOP technology®.•The iTOP technology® is a competitive delivery method when compared to electroporation and lipofection. The a...

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Bibliographic Details
Published in:	Journal of biotechnology Vol. 338; pp. 71 - 80
Main Authors:	Kholosy, Waleed M., Visscher, Marieke, Ogink, Kim, Buttstedt, Helen, Griffin, Kelli, Beier, Axel, Gerlach, Jan Patrick, Molenaar, Jan J., Geijsen, Niels, de Boer, Marco, Chatsisvili, Anna
Format:	Journal Article
Language:	English
Published:	Elsevier B.V 10-09-2021
Subjects:	CRISPR/Cas9 Gene editing hiPSCs iTOP Primary human T cells hiPSCs CRISPR/Cas9 iTOP Gene editing Primary human T cells
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Summary:	•The optimized iTOP technology® is a simple, fast and efficient intracellular delivery method for CRISPR/Cas9 RNPs.•Primary human cells can be edited successfully using the iTOP technology®.•The iTOP technology® is a competitive delivery method when compared to electroporation and lipofection. The advent of the CRISPR/Cas9 system has transformed the field of human genome engineering and has created new perspectives in the development of innovative cell therapies. However, the absence of a simple, fast and efficient delivery method of CRISPR/Cas9 into primary human cells has been limiting the progress of CRISPR/Cas9-based therapies. Here, we describe an optimized protocol for iTOP-mediated delivery of CRISPR/Cas9 in various human cells, including primary T cells, induced pluripotent stem cells (hiPSCs), Jurkat, ARPE-19 and HEK293 cells. We compare iTOP to other CRISPR/Cas9 delivery methods, such as electroporation and lipofection, and evaluate the corresponding gene-editing efficiencies and post-treatment cell viabilities. We demonstrate that the gene editing achieved by iTOP-mediated delivery of CRISPR/Cas9 is 40–95 % depending on the cell type, while post-iTOP cell viability remains high in the range of 70–95 %. Collectively, we present an optimized workflow for a simple, high-throughput and effective iTOP-mediated delivery of CRISPR/Cas9 to engineer difficult-to-transduce human cells. We believe that the iTOP technology® could contribute to the development of novel CRISPR/Cas9-based cell therapies.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0168-1656 1873-4863
DOI:	10.1016/j.jbiotec.2021.07.006