Prime Editing from PE1 to PE7: Advances in Precision Editing and Remaining Challenges in In Vivo Delivery
Background: Genome Editing Without Double-Strand Breaks
Prime editing is a genome-editing technology that enables targeted base substitutions, insertions, and deletions without DNA double-strand breaks or external donor DNA. A review in the June 2026 issue of BMB Reports summarizes the mechanism of this technology, first reported in 2019, improvements from PE1 to PE7, and strategies for large-scale editing and in vivo delivery. It reviews findings and limitations across multiple studies rather than reporting experiments on a newly developed editor.
Double-strand breaks caused by conventional CRISPR-Cas9 can lead to unwanted insertions or deletions, chromosomal rearrangements, or cytotoxicity. Prime editing instead nicks one DNA strand and uses reverse transcription to synthesize a new sequence. However, avoiding double-strand breaks by design does not eliminate all genotoxicity or off-target editing.
Key Findings: Joint Optimization of Enzymes, Guide RNA, and Repair Pathways
The basic system consists of a fusion protein combining a Cas9 nickase and reverse transcriptase, together with a prime editing guide RNA (pegRNA). After the Cas9 nickase nicks one DNA strand, the exposed end binds to the pegRNA’s primer-binding site. Reverse transcriptase copies the template contained in the pegRNA to synthesize new DNA. Editing is complete only after this sequence is incorporated and repair is finalized.
Each generation targets a different bottleneck. PE2 improves on PE1’s reverse transcriptase to enhance stability and DNA synthesis capacity. PE3 introduces an additional nick on the strand opposite the initially nicked strand to promote incorporation of the edited sequence, but it can also increase insertions and deletions. PE3b is designed so that the second nick occurs after editing, reducing this problem.
PE4 and PE5 transiently express dominant-negative MLH1, which interferes with the function of the normal protein, to suppress DNA mismatch repair (MMR). This reduces repair responses that restore the new sequence to its original state. Prolonged MMR suppression may compromise genome stability, so transient regulation is key. PE6 improved editor compactness and activity through protein engineering and directed evolution, while PE7 incorporated the RNA-binding domain of La protein into the editor by fusion to stabilize pegRNA.
Several strategies expand the range of edits. Twin prime editing (twin-PE) uses flaps—the overhanging portions of complementary new DNA strands generated by two pegRNAs—to change larger regions of sequence. PASTE, by contrast, uses an integrase, a DNA integration enzyme, to insert donor DNA at a recognition site installed by prime editing. The basic technology’s feature of requiring no donor DNA does not apply to every extension of prime editing.
Implications and Outlook: Distinguishing Delivery Methods and Stages of Application
For in vivo applications, delivery is as important as editing performance. Adeno-associated virus (AAV) has a packaging limit of about 4.7 kb, so strategies that split large editors for delivery are used. Persistent expression and immune responses are issues to consider. Engineered virus-like particles transiently deliver editor protein–RNA complexes, while lipid nanoparticles (LNPs) carry editor messenger RNA, pegRNA, and other components. The review examines these as distinct delivery strategies.
As an example of clinical application, the review describes an early report of ex vivo prime editing of autologous CD34+ hematopoietic stem cells from a patient with chronic granulomatous disease (CGD). In vivo correction in the retina, brain, and liver is presented as preclinical work, including studies in mice. The existence of an early clinical report alone is not enough to establish long-term safety or the durability of therapeutic benefit.
Remaining challenges include differences in efficiency across tissues and cell types, pegRNA instability, off-target editing, immune responses, and delivery costs. Rather than choosing an editor based only on its generation number, efficiency, unwanted editing products, and safety should be compared for the target mutation and delivery conditions. Technology trends and application examples are summarized in the full BMB Reports review.
Nature Biotechnology, Published June 2026.
Summary: Bypassing the intrinsic double-strand break (DSB) liabilities and random indel mutations that inherently bottleneck conventional wild-type CRISPR-Cas9 pipelines, this comprehensive review systemizes the structural translation of Prime Editing (PE) architectures. Operating via a high-performance fusion protein composed of an engineered Cas9 nickase and an optimized reverse transcriptase matched with a prime editing guide RNA (pegRNA), the platform executes search-and-replace edits including precise base substitutions, scarless insertions, and deletions. Tracing the methodological breakthroughs from PE1 up to the hyper-efficient PE7 system, the framework captures the kinetics of twin-PE large-fragment genomic integration alongside non-integrating lipid nanoparticle (LNP) delivery vectors, establishing a predictive, high-throughput computational baseline for universal patient stratification and targeted clinical translation.
Developing prime editing as a therapy requires testing both its ability to change a mutation and its ability to reach the cells that need treatment. For ex vivo correction of a patient’s hematopoietic stem cells, evaluation should include correction rates, unwanted insertions and deletions, off-target editing, and cell function. For direct in vivo correction in the retina or liver, delivery to the target tissue and the duration of editor expression also need to be assessed. This review summarizes the technical evidence and constraints to consider when selecting an editor, pegRNA, and delivery vehicle together. Early clinical reports and preclinical results are starting points for further evaluation; they do not establish long-term safety or guarantee successful manufacturing and regulatory approval.