Efficient Gene Editing at Major CFTR Mutation Loci.

Area of Science:

• Genetics
• Molecular Biology
• Regenerative Medicine

Background:

• Cystic Fibrosis (CF) is a lethal autosomal recessive genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
• Nuclease-mediated precise gene editing (PGE) is a potential therapeutic strategy for CF, but requires efficient, viral vector-free (VDR-free) delivery methods.

Purpose of the Study:

• To compare different transfection methods and formats for delivering CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) for precise gene editing of CFTR mutations.
• To establish an efficient VDR-free gene editing strategy for CF therapy.

Main Methods:

• Comparison of transfection methods (lipofectamine vs. electroporation) and formats (plasmid DNA vs. ribonucleoprotein) for CRISPR/Cas9 delivery.
• Utilized CRISPR/Cas9 ribonucleoprotein (Cas9 RNP) complexes with ssODNs to target CFTR mutation loci in induced pluripotent stem cells (iPSCs).
• Assessed gene editing efficiency and CFTR function restoration in iPSC-derived lung organoids and CFPAC-1 cells.

Main Results:

• Electroporation of Cas9 RNP demonstrated superior efficiency compared to other tested methods.
• Achieved 4.8% to 27.2% editing efficiency for dF508, G542X, and G551D mutations in wild-type iPSCs.
• Demonstrated over 20% precise correction rate in a patient-derived iPSC line and restored CFTR function in relevant cellular models.

Conclusions:

• Electroporation of CRISPR/Cas9 RNP is an effective VDR-free method for precise gene editing of CFTR mutations.
• This approach shows feasibility for developing gene editing-based therapeutics for monogenic diseases like CF.
• Restoration of CFTR function in organoids and cell lines validates the therapeutic potential of this gene editing strategy.