Prime Editing Modification with FEN1 Improves F508del Variant Editing in the CFTR Gene in Airway Basal Cells

Area of Science:

• Gene editing technologies
• Molecular biology
• Genetic disease therapies

Background:

• Prime editing offers a potential strategy for correcting disease-causing genetic mutations.
• The efficiency of prime editing varies depending on the specific genomic location and sequence context.
• The CFTR F508del variant is a common cause of cystic fibrosis, necessitating effective gene correction strategies.

Purpose of the Study:

• To systematically evaluate modifications of the PEmax prime editing system for enhanced efficiency.
• To assess the performance of prime editing variants in correcting the CFTR F508del pathogenic variant in patient-derived cells.
• To identify prime editor modifications that can overcome challenges in AT-rich genomic regions.

Main Methods:

• Systematic evaluation of 12 PEmax system modifications.
• Utilized EXO1 and FEN1 nucleases to enhance the prime editing system.
• Tested editing efficiency in patient-derived airway basal cells harboring the CFTR F508del variant.

Main Results:

• All tested prime editor variants exhibited low efficiency in the AT-rich target region.
• The 4-FEN modification demonstrated a significant improvement in editing rates, up to 2.13-fold higher than standard PEmax.
• Despite improvements, AT-rich sequences present a persistent challenge for prime editing.

Conclusions:

• Optimized prime editing systems, such as 4-FEN, can enhance editing efficiency for specific pathogenic variants.
• The 4-FEN modification shows potential for broader applications in correcting other genomic targets.
• Further development is needed to address the inherent difficulties in editing AT-rich genomic sequences.