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

Kholosy, W.M.; Visscher, M.; Ogink, K.; Buttstedt, H.; Griffin, K.; Beier, A.; Gerlach, J.P.; Molenaar, J.J.; Geijsen, N.; Boer, M. de; Chatsisvili, A.

doi:doi:10.1016/j.jbiotec.2021.07.006

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...Show moreThe 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 (R) could contribute to the development of novel CRISPR/Cas9-based cell therapies.Show less

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