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.

CRISPR/Cas9系统的出现改变了人类基因组工程领域，为创新细胞疗法的发展开辟了新的视角。然而，缺乏简单、快速和有效的 CRISPR/Cas9 递送方法到原代人类细胞一直限制了基于 CRISPR/Cas9 的疗法的进展。在这里，我们描述了在各种人类细胞中 iTOP 介导的 CRISPR/Cas9 递送的优化方案，包括原代 T 细胞、诱导多能干细胞 (hiPSC)、Jurkat、ARPE-19 和 HEK293 细胞。我们将 iTOP 与其他 CRISPR/Cas9 递送方法（例如电穿孔和脂质转染）进行比较，并评估相应的基因编辑效率和处理后细胞活力。我们证明 iTOP 介导的 CRISPR/Cas9 传递实现的基因编辑是 40-95%，具体取决于细胞类型，而 iTOP 后细胞活力保持在 70-95% 的范围内。总的来说，我们提出了一个优化的工作流程，用于简单、高通量和有效的 iTOP 介导的 CRISPR/Cas9 传递，以设计难以转导的人类细胞。我们相信 iTOP 技术® 有助于开发基于 CRISPR/Cas9 的新型细胞疗法。