A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells

Cuevas-Ocaña, Sara; Yang, Jin Ye; Aushev, Magomet; Schlossmacher, George; Bear, Christine E.; Hannan, Nicholas R.F.; Perkins, Neil D.; Rossant, Janet; Wong, Amy P.; Gray, Michael A.

Abstract

Introducing or correcting disease-causing mutations through genome editing in human pluripotent stem cells (hPSCs) followed by tissue-specific differentiation provide sustainable models of multiorgan diseases, such as cystic fibrosis (CF). However, low editing efficiency resulting in extended cell culture periods and the use of specialised equipment for fluorescence activated cell sorting (FACS) make hPSC genome editing still challenging. We aimed to investigate whether a combination of cell cycle synchronisation, single-stranded oligodeoxyribonucleotides, transient selection, manual clonal isolation, and rapid screening can improve the generation of correctly modified hPSCs. Here, we introduced the most common CF mutation, ΔF508, into the CFTR gene, using TALENs into hPSCs, and corrected the W1282X mutation using CRISPR-Cas9, in human-induced PSCs. This relatively simple method achieved up to 10% efficiency without the need for FACS, generating heterozygous and homozygous gene edited hPSCs within 3–6 weeks in order to understand genetic determinants of disease and precision medicine.

Citation

Cuevas-Ocaña, S., Yang, J. Y., Aushev, M., Schlossmacher, G., Bear, C. E., Hannan, N. R., …Gray, M. A. (2023). A Cell-Based Optimised Approach for Rapid and Efficient Gene Editing of Human Pluripotent Stem Cells. International Journal of Molecular Sciences, 24(12), Article 10266. https://doi.org/10.3390/ijms241210266

• Journal Article Type: Article
• Acceptance Date: Jun 9, 2023
• Online Publication Date: Jun 17, 2023
• Publication Date: Jun 17, 2023
• Deposit Date: Jun 20, 2023
• Publicly Available Date: Jun 20, 2023
• Journal: International Journal of Molecular Sciences
• Print ISSN: 1661-6596
• Electronic ISSN: 1422-0067
• Publisher: MDPI
• Peer Reviewed: Peer Reviewed
• Volume: 24
• Issue: 12
• Article Number: 10266
• DOI: https://doi.org/10.3390/ijms241210266
• Keywords: stem cells; gene editing; TALEN; CRISPR-Cas9; ΔF508; W1282X; cystic fibrosis
• Public URL: https://nottingham-repository.worktribe.com/output/22176332
• Publisher URL: https://www.mdpi.com/1422-0067/24/12/10266