The past two decades have seen the growing development and consequent vast application of next-generation genome editing tools in fundamental and applied research. Nowadays GE based on RNA-guided... Show moreThe past two decades have seen the growing development and consequent vast application of next-generation genome editing tools in fundamental and applied research. Nowadays GE based on RNA-guided nucleases (e.g., engineered CRISPR-Cas9 nucleases) are the most common tools for targeted genetic modification. Nevertheless, these technologies are in need of increased efficiency and accuracy, especially looking forward to translation into diverse clinical applications. The work presented in this thesis focuses on improving the efficiency and accuracy of genome editing, particularly in cells with high therapeutic potential, such as induced pluripotent stem cells (iPSCs), by investigating the feasibility of using adenoviral vectors to test novel genome editing approaches and by exploring the possible applications of a scarless strategy. Show less
Kholosy, W.M.; Visscher, M.; Ogink, K.; Buttstedt, H.; Griffin, K.; Beier, A.; ... ; Chatsisvili, A. 2021
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... 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
Holkers, M.; Cathomen, T.; Goncalves, M.A.F.V. 2014
