Objective:

Establish a fast, efficient, and reliable genome editing pipeline for induced pluripotent stem cells (iPSCs), enabling the generation of isogenic cell lines for disease modeling and functional studies.

Methods:

iPSCs were reprogrammed from both patient-derived and healthy donor cells. To eliminate confounding effects of genetic background in comparative studies, CRISPR/Cas9 genome editing was employed to either correct pathogenic mutations in patient-derived iPSCs or introduce specific mutations into healthy donor iPSCs. We optimized in particular the stoichiometry of the Cas9 protein, guide RNA (gRNA), and single-stranded oligodeoxynucleotide (ssODN) to improve editing efficiency. A series of quality control steps, including genotyping and off-target analysis, were implemented to ensure the fidelity of edited lines.

Results:

The optimized Cas9:gRNA:ssODN ratio significantly enhanced genome editing efficiency. Quality control procedures confirmed successful generation of isogenic iPSC lines with minimal off-target effects. Despite initial challenges in establishing the editing pipeline, the support from the CorEuStem network, particularly the core facilities at CRG (Barcelona) and MDC (Berlin), was instrumental in overcoming technical hurdles and accelerating workflow implementation.

Conclusion:

We successfully established a robust and efficient genome editing workflow within our iPSC core facility. This pipeline enables the generation of isogenic cell lines, facilitating more accurate functional comparisons and disease modeling. Collaborative support networks played a critical role in accelerating protocol optimization and ensuring reproducibility.