Simultaneous high-efficiency base editing and reprogramming of patient fibroblasts

Area of Science:

• Stem cell biology
• Genetic engineering
• Molecular medicine

Background:

• Human induced pluripotent stem cells (hiPSCs) are crucial for studying genetic diseases and developing personalized therapies.
• Gene editing technologies, particularly CRISPR-Cas9, offer precise modification of targeted DNA loci for various hiPSC applications.
• Monogenic diseases benefit from gene editing to understand mutation functions and generate corrected patient-derived hiPSCs.

Purpose of the Study:

• To develop a highly efficient method for simultaneous base editing and reprogramming of fibroblasts into hiPSCs.
• To demonstrate the application of this method for generating gene-edited hiPSCs from patients with specific monogenic diseases.
• To confirm the restoration of gene function in corrected hiPSCs.

Main Methods:

• Employing a CRISPR-Cas9 adenine base editor for simultaneous base editing and reprogramming of fibroblasts.
• Utilizing skin biopsies from four patients with Finnish-founder pathogenic point mutations in NOTCH3 or LDLR genes.
• Generating and characterizing gene-edited hiPSC monoclonal lines.

Main Results:

• Achieved high efficiency in generating tens of gene-edited hiPSC monoclonal lines.
• Successfully created gene-edited hiPSCs from patients with NOTCH3 and LDLR mutations.
• Demonstrated restoration of LDLR activity following gene correction in hiPSCs.

Conclusions:

• The described method offers an efficient and robust approach for generating gene-edited hiPSCs.
• This technique significantly reduces cell culture time, minimizing risks of in vitro alterations.
• The approach holds promise for advancing genetic disease research and personalized stem cell therapies.