Background

Conventional gene editing technology, CRISPR/Cas9, has raised safety concerns due to its induction of double-strand breaks (DSBs) in DNA, potentially causing genome damage. Particularly, the repair process in cells can lead to fatal genome damage such as random base mutations or chromosomal translocations. To overcome these side effects, Prime Editing (PE) was developed, successfully enabling the precise addition or deletion of small genetic information without cutting the DNA double helix.

However, PE also has clear limitations to overcome. Due to its reliance on reverse transcription, it is useful for inserting small-scale sequences of dozens of base pairs (bp), but its efficiency decreases when inserting large genes of several kilobase pairs (kbp) required for disease treatment. The academic community has previously considered combining PE with recombinase to insert large genes. This method, however, is known to increase the complexity of therapeutic development by leaving unwanted recombinant sequences in the genome or requiring the delivery of multiple proteins into cells.

Key Discovery

Precision Insertion of Large Genes via DNA Flap Binding

A research team led by Professor Bae Sangsu from the School of Medicine at Seoul National University successfully overcame these limitations by enhancing PE. The team's proposed solution is the Prime Assembly (PA) technology, which induces complementary single-stranded 3' flaps at both the genomic target site and the donor DNA, allowing them to hybridize. PA is the result of applying the in vitro DNA assembly principle of Gibson Assembly to intracellular gene editing.

To optimize the efficiency of PA, the research team designed three methods: Single-Flap (SF)-PA, SFn-PA (SF-PA with an additional nick on the opposite strand), and Dual-Flap (DF)-PA (with two pairs of 3' flaps on both sides for hybridization), and compared their performance. In targeted gene replacement experiments using human embryonic kidney cells (HEK293T), DF-PA demonstrated a maximum efficiency of 57.8% in inserting a 2.9kbp donor DNA gene fragment. The sequence accuracy of the inserted gene fragment also exceeded 99%, confirming its precision.

Conventional insertion methods based on homology-directed repair (HDR) have limitations in that they only work during active cell division, making them difficult to apply to non-dividing cells. In contrast, PA technology operates independently of the cell cycle, showing high efficiency regardless of cell division status.

Significance and Prospects

A Foundation for Universal Gene Therapy Development

This study has drawn academic interest for its ability to achieve large-scale gene replacement while avoiding the inherent drawbacks of CRISPR gene scissors, such as off-target effects and chromosomal abnormalities. It is expected to be highly useful in treating rare diseases such as hemophilia and hereditary retinal diseases, where various mutations occur randomly within the gene. The study demonstrated the validity of a treatment strategy that replaces the entire damaged region with a normal sequence without the need to create a custom editor for each patient's mutation. If such a universal treatment strategy becomes a reality, the efficiency of future gene therapy development is likely to increase significantly.

However, to apply this technology to actual patient treatment, the limitations of delivery methods must first be resolved. The development of high-capacity delivery systems capable of transporting large genes and PA proteins into target cells is a key challenge. Research is currently underway to improve the delivery efficiency of existing vectors such as adeno-associated virus (AAV) and lipid nanoparticles (LNP). The research team expressed their intention to focus on verifying in vivo safety using animal models in the future.