Enhancing prime editing by fusing polymerase substrate-binding proteins to reverse transcriptase.

Area of Science:

• Molecular Biology
• Gene Editing Technologies

Background:

• Prime editing (PE) allows precise small DNA modifications but has limited efficiency in human cells.
• The reverse transcriptase (RT) component's activity is crucial for PE performance.

Purpose of the Study:

• To enhance prime editing efficiency by fusing a polymerase substrate binding protein (PSBP) with the reverse transcriptase (RT).
• To identify specific PSBP modules that improve PE outcomes.

Main Methods:

• A twelve-member screen of PSBP fusions with RT was conducted.
• The NHP6A HMG box module was identified as a lead fusion candidate.
• Compatibility with existing PE systems (PE3, PE4, PE7) and clinically relevant alleles was tested.

Main Results:

• NHP6A-RT fusion significantly increased intended edits (substitutions, insertions, deletions) with low indel formation.
• The PSBP fusion enhanced editing across various PE systems, including PE3, PE4, and PE7.
• Four clinically relevant alleles were efficiently installed with high product purity using the NHP6A-La combination.

Conclusions:

• PSBP fusion is a viable strategy to enhance prime editing efficiency and precision.
• Compact accessory modules like PSBP can be integrated into next-generation prime editing platforms.
• The NHP6A-RT fusion offers a precise method for improving prime editing outcomes in therapeutic applications.