Precise Genome Editing without Double-Strand Cleavage: Recent Advances and Challenges in Prime Editing for Therapeutic Applications
Background
Genome editing (GE) technology has rapidly advanced since the emergence of CRISPR-Cas9. Existing technologies involve double-strand DNA cleavage, which carries the risk of unintended genomic alterations. Prime editing (PE), designed to address this, is a precise editing platform that combines reverse transcriptase and a prime editing guide RNA (pegRNA). It can modify target sequences without double-strand breaks (DSB), making it highly safe.
Early PE models demonstrated the potential for genome editing, but their efficiency was limited by interference from cellular mismatch repair (MMR) responses and the influence of chromatin structure. In particular, when inserting large gene fragments, the editing rate was observed to decrease sharply. This review summarizes the results of pegRNA design structure, DNA flap dynamics, and repair pathway interaction mechanisms in a clear and concise manner. Furthermore, it analyzes and presents the working principles and development challenges of improved systems with enhanced efficiency.
Key Findings
Protein Engineering and Cellular Repair Control
The core of prime editing is the stable expression of the complex. The researchers designed the PEmax system, which improves the protein structure and carefully adjusts the placement of the nuclear localization signal (NLS) and codon sequence. This design exhibits enhanced editing activity in human cells. In addition, PE4 and PE5 were developed to suppress the interference phenomenon in which the cellular MMR mechanism recognizes the intermediate (DNA flap) as damage and removes it. A key feature is the overexpression of a dominant-negative form of the MLH1 gene to inhibit the MMR pathway, thereby significantly increasing editing efficiency.
Large-Scale Gene Insertion Technology
Existing PE has the limitation of being restricted to small-scale editing. To overcome this, the researchers constructed the TWIN-PE, PASTE, and PrimeRoot platforms. TWIN-PE induces two pegRNAs to edit each strand of the DNA, forming complementary flap binding. This method successfully achieves the inversion or deletion of sequences of hundreds of base pairs. Meanwhile, PASTE and PrimeRoot are systems that fuse site-specific recombinase with PE. They insert a small recognition sequence into the genome and then introduce the recombinase to accurately transplant foreign genes of up to 10 kilobases (kb) into the desired location, demonstrating high efficiency.
Significance and Prospects
The refined PE research summarized in this study is expected to be useful in various fields, including the development of treatments for genetic diseases, crop breeding, and microbial engineering. However, there are still significant barriers to entry for clinical treatments or actual applications in agriculture. The stereochemical limitations of the chromatin where the target gene is located and the variation in MMR efficiency between cell types are challenges that need to be overcome. Optimizing the delivery system for transporting the PE complex to cells in vivo is a crucial requirement. Furthermore, the technology needs to be improved to completely control the micro-mutations that occur during the insertion of large DNA fragments in order to safely enter the commercialization stage.
Prime editing has become a highly programmable and accurate genome-editing platform that can install targeted substitutions, insertions, and deletions without introducing double-strand breaks or requiring a separate donor DNA template. This review summarizes recent developments about prime editing mechanisms, such as knowledge about flap dynamics, repair pathway interactions, and pegRNA architecture, and improvements in engineering, resulting in high-efficiency systems, including PEmax, PE4/5, TWIN-PE, PASTE, and PrimeRoot. Such advances now make prime editing applicable to therapeutic gene correction, agricultural biotechnology, microbial engineering, and functional genomics. However, delivery, chromatin context, mismatch-repair variability, and large-fragment integration remain major barriers to broad application. By comparing prime editing with other genome-editing modalities, this review summarizes its unique advantages and highlights strategic innovations needed for its next stage of development. Together, these developments position prime editing as a highly programmable platform with strong potential to shape the future of precise genome rewriting.
This research provides a practical turning point for the development of personalized treatments for patients with rare genetic diseases. By overcoming the genomic instability caused by existing gene editing tools, it maximizes the potential for treating diseases that require large-scale gene editing, such as Duchenne Muscular Dystrophy (DMD) and Hemophilia. Specifically, the PASTE technology can be used to completely replace the damaged gene sequence in the patient's hepatocytes or muscle cells with a normal gene sequence.
In the agricultural biotechnology industry, it is expected to accelerate the development of non-transgenic new crop varieties by improving crop traits without introducing foreign DNA. It is expected to contribute to securing food resources by breeding rice or soybean varieties with complex environmental stress resistance in a short period of time, thereby responding to climate change.