A revolutionary gene-editing tool is making waves, offering the promise of correcting disease-causing mutations with pinpoint accuracy. But what happens when this sophisticated technology meets the complex biology of a living animal?
The discovery of CRISPR-Cas9 revolutionized genetic engineering, earning its pioneers the Nobel Prize in 2020. Yet, this powerful tool often cuts DNA unpredictably, leading to unintended mutations that limit its therapeutic potential3 .
Enter prime editing—a more precise "search-and-replace" gene-editing system that directly writes new genetic information into a target DNA sequence without causing double-stranded breaks. While exceptionally promising, the journey of implementing this technology in living organisms like mice has revealed both impressive capabilities and significant challenges, particularly around editing efficiency and unwanted byproducts.
Traditional CRISPR-Cas9 gene editing works like molecular scissors, cutting both strands of the DNA helix at specific locations. This process relies on the cell's own repair mechanisms to fix the break, which can be error-prone and lead to a mix of desired and random, unwanted edits3 .
Prime editing represents a monumental leap forward. Think of it not as scissors, but as a word processor with a "find-and-replace" function for DNA. This sophisticated system can theoretically correct about 89% of known disease-causing genetic variations in humans1 4 .
| Editor | Key Improvements | Editing Efficiency |
|---|---|---|
| PE1 | Original proof-of-concept | ~10-20% in human cells |
| PE2 | Engineered reverse transcriptase | ~20-40% in human cells |
| PE3 | Additional sgRNA to nick non-edited strand | ~30-50% in human cells |
| PEmax | Optimized nuclear localization and expression | 1.5-2.1 fold improvement over PE25 |
| PE6 series | Evolved, compact reverse transcriptases | Up to ~70-90% in human cells |
Initial proof-of-concept demonstrating prime editing feasibility
Improved reverse transcriptase for better efficiency
Dual guide system to enhance editing rates
Optimized for in vivo applications
Compact, highly efficient editors with improved delivery
In 2021, a pivotal study demonstrated prime editing's functionality in adult mice for the first time, marking a critical step toward therapeutic applications5 . Researchers faced the formidable challenge of delivering the relatively large prime editing machinery into mouse cells efficiently.
Created PE2* by adding specific nuclear localization signals to ensure the prime editor efficiently reached its DNA target in the nucleus. This modification improved nuclear localization from approximately 60% to nearly 100% in some cell types5 .
Since the prime editing system is too large to fit into a single adeno-associated virus (AAV)—a common gene therapy delivery vehicle—they split the editor into two parts, each delivered by a separate AAV vector. These parts reassembled inside the target cells using a technique called split intein-mediated protein splicing5 .
Prime editing successfully corrected the disease-causing mutation in the mouse liver, showing the potential for therapeutic application.
Editing efficiency remained relatively low, with unwanted indels (insertions/deletions) appearing as byproducts of the editing process5 .
The quantitative results from this and subsequent studies reveal the current state of prime editing in animal models:
Key Insight: The data reveals a crucial trade-off: while prime editing offers superior precision in the types of changes it can make, its efficiency in living animals currently lags behind both traditional CRISPR and base editing technologies.
Since those early in vivo experiments, researchers have developed numerous innovations to address the challenges of efficiency and unwanted byproducts:
| Research Solution | Function | Impact |
|---|---|---|
| epegRNAs | Engineered pegRNAs with stabilizing RNA motifs | 3-4 fold efficiency improvement by reducing degradation1 |
| Dual AAV System | Split editor delivered via two AAV vectors | Enables in vivo delivery despite large size5 |
| PE6 Series | Evolved, compact reverse transcriptases | Higher efficiency and smaller size for improved delivery4 |
| ProPE | Separates nicking and template functions | Expands editing window and enhances efficiency8 |
| PE-tag | Method to identify off-target sites | Improves safety profiling9 |
Novel viral and non-viral delivery systems to transport prime editors more efficiently
Using techniques like PACE to create more efficient and compact reverse transcriptases4
Studying cellular processes that resolve edited DNA flaps to favor desired outcomes4
The journey of prime editing from petri dishes to living organisms represents both a remarkable achievement and a work in progress. While the induction of unwanted outcomes in early mouse studies highlighted real challenges, the scientific community has responded with innovative solutions that continue to improve the technology's precision and efficiency.
The initial "undesired outcomes" observed in mice represent not dead ends, but rather necessary stepping stones in the rigorous scientific process of translating a revolutionary technology from concept to clinic.
The future of genetic medicine may well depend on our ability to precisely edit the fundamental code of life, and prime editing—despite its current limitations—remains one of our most promising tools for achieving this goal.