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

Autor: Kholosy WM; Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, the Netherlands., Visscher M; NTrans Technologies BV, Heidelberglaan 7, 3584 CS Utrecht, the Netherlands., Ogink K; NTrans Technologies BV, Heidelberglaan 7, 3584 CS Utrecht, the Netherlands., Buttstedt H; NTrans Technologies BV, Heidelberglaan 7, 3584 CS Utrecht, the Netherlands., Griffin K; NTrans Technologies BV, Heidelberglaan 7, 3584 CS Utrecht, the Netherlands., Beier A; KNAW-Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands., Gerlach JP; NTrans Technologies BV, Heidelberglaan 7, 3584 CS Utrecht, the Netherlands., Molenaar JJ; Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, the Netherlands., Geijsen N; KNAW-Hubrecht Institute, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands., de Boer M; NTrans Technologies BV, Heidelberglaan 7, 3584 CS Utrecht, the Netherlands., Chatsisvili A; NTrans Technologies BV, Heidelberglaan 7, 3584 CS Utrecht, the Netherlands. Electronic address: a.chatsisvili@uu.nl.
Jazyk: angličtina
Zdroj: Journal of biotechnology [J Biotechnol] 2021 Sep 10; Vol. 338, pp. 71-80. Date of Electronic Publication: 2021 Jul 13.
DOI: 10.1016/j.jbiotec.2021.07.006
Abstrakt: 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.
(Copyright © 2021 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
Externí odkaz: