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

Waleed M. Kholosy, Marieke Visscher, Kim Ogink, Helen Buttstedt, Kelli Griffin, Axel Beier, Jan Patrick Gerlach, Jan J. Molenaar, Niels Geijsen, Marco de Boer, Anna Chatsisvili

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)71-80
Number of pages10
JournalJournal of Biotechnology
Volume338
DOIs
Publication statusPublished - 10 Sept 2021

Keywords

  • CRISPR/Cas9
  • Gene editing
  • hiPSCs
  • iTOP
  • Primary human T cells

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