How can gene-editing of human pluripotent stem cells help cystic fibrosis?

Cystic fibrosis (CF) is a monogenic recessive disorder, affecting 70,000 people worldwide. CF is due to mutations in the CFTR gene, resulting in a defective protein that leads to symptoms in numerous organs, especially lungs. Despite recent advances, the lack of CF material contributes to the unmet need to find effective treatments for 55% of CF patients, and to restore the function of all affected tissues. A potential strategy would be to use gene-editing technologies (TALENs or CRISPR/Cas9) to introduce into or correct specific mutations in pluripotent stem cells (hPSCs) that can be turned into any other cell type. But current protocols for gene-editing hPSCs are often complicated, lengthy (6-8 months) and costly, thus, being restricted to a few specialised labs. This study describes an optimised protocol to correct the W1282X mutation in 3 CF hPSC lines derived from an adult and children with CF (iPSCs), by transfecting a CRISPR/Cas9 plasmid, a selection plasmid and a donor template. Corrected iPSCs were produced in 3-6 weeks. This protocol was previously used to generate the most common CF genotype, ∆F508/∆F508, from healthy hPSCs using TALENs. This work demonstrated that optimising each step of the gene-editing process, including an efficient transfection of hPSCs, and rapid isolation and screening for corrected cells, can produce in vitro models for CF in less than one month with minimum costs. These cells generated from paediatric or adult CF patients can be used to generate mutation-customised tissue-specific cultures holding great promise to advance drug testing and personalised medicine for CF. Supported by CF Trust-SRC003, CFIT (SickKids, CF-Canada Foundation), Emily’s Entourage.