Optimized nickase- and nuclease-based prime editing in human and mouse cells

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Prime editing (PE) offers precise genomic modification for research and clinical use.
• Existing PE systems face efficiency challenges in certain cell types.

Purpose of the Study:

• To develop and evaluate all-in-one prime editing constructs (PEA1).
• To enhance prime editing efficiency in difficult-to-edit cell lines (K562, HeLa).
• To assess the efficacy of a nuclease prime editor in vitro and in vivo.

Main Methods:

• Generation of all-in-one prime editing (PEA1) constructs with selection markers.
• Testing PEA1 constructs in HEK293T, K562, HeLa, and mouse embryonic stem (ES) cells.
• Development and testing of a nuclease prime editor in cell lines and mouse embryos via zygotic injection.

Main Results:

• PEA1 constructs showed high efficiency (up to 95%) in HEK293T cells but low efficiency in K562 and HeLa cells.
• The nuclease prime editor increased initiation but caused extra insertions in K562, HeLa, and mouse ES cells.
• Zygotic injection of the nuclease prime editor resulted in up to 100% correct modifications in mouse fetuses.

Conclusions:

• PEA1 constructs are effective in some cell types but require optimization for others.
• Nuclease prime editors show promise for in vivo applications, particularly in mouse models.
• Further refinement is needed to minimize off-target edits and improve repair template utilization.