Prime editing with reverse transcriptase demonstrates the potential for correcting mutations in non-dividing neurons
Background
Conventional CRISPR-Cas9 gene editing technology relies on creating double-strand breaks in DNA. This process can trigger unwanted insertions or deletions due to the cell's own repair mechanisms, and also raises concerns about genomic damage and toxicity. In non-dividing cells, such as neurons, DNA repair activity is low, making them particularly vulnerable to permanent cell death or mutations when using traditional cleavage-based techniques. Since most neurological disorders are caused by subtle single-base mutations or deletions, there is a critical need for precise genome editing techniques that can accurately correct these defects.
Key Findings
Prime editing (PE) is an advanced gene editing technology that allows for the precise insertion, deletion, or substitution of genetic information without creating double-strand breaks. This editing tool consists of a Cas9 nickase fused with a reverse transcriptase (RT) and a prime editing guide RNA (pegRNA). The pegRNA targets the desired sequence and provides a template for the corrected sequence. The fused protein then performs a reverse transcription reaction, precisely replacing the target DNA sequence.
However, when prime editing is performed in fully differentiated adult neurons, the editing efficiency is less than 10%, indicating a need for improvement. Furthermore, the complex structure of the pegRNA molecule can lead to its degradation within cells and increase the risk of off-target errors during the reverse transcription process.
To overcome these limitations, researchers have developed various engineering solutions. One approach is the use of split-adeno-associated virus (split-AAV) systems, which divide the prime editor protein into two parts to increase delivery efficiency. Another approach involves conjugating engineered peptides that target neurons to the surface of lipid nanoparticles (LNPs) to enhance their ability to cross the blood-brain barrier (BBB). Additionally, the use of smaller Cas variants has been explored to overcome payload limitations, and guide RNA engineering has been used to improve editing efficiency in neurons by more than three times.
Significance and Outlook
These genetic engineering improvements have led to promising results in preclinical models. In cell models of fragile X syndrome and other monogenic neurodevelopmental disorders, as well as in mouse studies, prime editing has successfully corrected genetic defects, induced normal protein expression, and alleviated disease symptoms.
However, there are still challenges to overcome before prime editing can be used clinically. One concern is the potential for off-target mutations caused by the long-term presence of the editor protein in brain tissue. Therefore, it is important to develop transient expression systems that allow the editor protein to function for a limited time and then disappear. Additionally, it is essential to establish a robust evaluation protocol that uses whole-genome sequencing to ensure the clinical safety of prime editing before it is used in patients. In the future, if targeted cell selection and drug delivery efficiency can be improved, prime editing could offer a completely new treatment option for patients with previously untreatable neurological and psychiatric disorders.
Prime editing, a novel clustered regularly interspaced short palindromic repeats (CRISPR)-based technology, fuses a reverse transcriptase (RT) to an engineered CRISPR-associated protein 9 (Cas9) and uses a prime editing guide RNA (pegRNA)-encoded template. It enables precise base substitutions, small insertions, and deletions without introducing double-strand breaks, thereby expanding the range of correctable mutations while reducing undesired repair outcomes. This technology offers a promising strategy for genomic correction in the nervous system. Here, we review the development of prime editing, its mechanistic rationale, and emerging preclinical evidence that supports its application in neuropsychiatric disorders. We discuss key biological and technological barriers, including limited editing efficiency in post-mitotic neurons, complex pegRNA design, reverse transcription-related errors, vector payload limitations, and blood-brain barrier (BBB) penetration. Nevertheless, in vitro and in vivo studies have demonstrated proof-of-concept correction and functional rescue in several monogenic neurodevelopmental disorders. Advances such as split-adeno-associated virus (AAV) systems, lipid nanoparticles, engineered peptides, and compact Cas variants are actively expanding their therapeutic potential. Further clinical translation will rely on improved editors with guide engineering, BBB-penetrant and neuron-targeted delivery platforms, transient or cell-type-specific expression strategies, and comprehensive genome-wide safety evaluations.
The results of this study can lead to the development of a therapeutic strategy for directly correcting repeat sequence mutations in the striatal neurons of Huntington's disease patients. One possible scenario is the injection of LNPs coated with a targeting peptide into the cerebrospinal fluid of patients, which would allow for the localized correction of genes in the affected neurons. From an industrial perspective, the development of prime editing is likely to drive the growth of the market for artificial intelligence (AI)-based software that can assist with the design of complex pegRNAs. Furthermore, collaborations between platform biotechnology companies and global pharmaceutical companies are expected to increase as they seek to overcome the longstanding challenges of in vivo delivery of gene therapies.