Gene-editing of human pluripotent stem cells for cystic fibrosis modelling

Abstract

Cystic fibrosis (CF) is a genetic disorder caused by mutations in the CFTR gene. A defective CFTR protein produces an impaired ion and fluid secretion in the epithelial cells that affects many organs and leads to severe lung disease. Despite recent treatments have been approved for specific genotypes, ~ 44% of people with CF still lack an effective treatment. Therefore, there is a need for more CF research and drug testing in order to find treatments for all CF patients and all relevant affected tissues. Human pluripotent stem cells (hPSCs) hold the potential to provide unlimited production of tissue-specific in vitro live cultures. Geneediting technologies, including TALENs and CRISPR/Cas9 systems, enable introducing or correcting specific mutations for disease modelling, although this is still challenging and time-consuming in hPSCs. This project demonstrates the generation of a robust hPSC geneediting strategy that can be performed in a relatively short time in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) without specialised equipment. This strategy consists of (i) the efficient transfection of engineered nucleases, ssODN and a plasmid containing puromycin resistance into S phase pre-synchronised hPSCs. (ii) Rapid assessment of nuclease activity and integration or correction of the mutation of interest in the initial mixed population, (iii) followed by a rapid isolation and identification of correctly modified clones. This strategy was used to generate a homozygous ΔF508-CF hESC line, and to correct W1282X-CF in three different iPSC lines, with 1-10% efficiency, within 3 weeks. These gene-edited cells hold great potential for the CF research, CF drug testing and CF personalise medicine fields