A genome editing platform for enhancing prime editing efficiency through pegRNA stabilization and protection

Background

Genome editing has revolutionized the field of molecular biology and medicine by enabling precise alterations to DNA sequences within living cells. Among the most promising genome editing technologies is prime editing, a CRISPR-based approach that allows for targeted insertions, deletions, and base substitutions without introducing double-stranded DNA breaks. Prime editing relies on a fusion protein composed of a Cas9 nickase and a reverse transcriptase, guided by a specialized RNA molecule known as the prime editing guide RNA (pegRNA). This technology holds immense potential for correcting disease-causing mutations, creating animal models, and advancing gene therapy. However, the efficiency and reliability of prime editing are often hampered by technical challenges, particularly those related to the stability and integrity of the pegRNA molecule.

Current approaches to prime editing frequently encounter limitations due to the rapid degradation of pegRNAs, especially at their 3’ ends, which contain critical sequences for DNA targeting and synthesis. Exonuclease-mediated degradation of the 3’ extension of pegRNAs reduces the availability of functional guide RNAs, leading to suboptimal editing efficiencies and inconsistent outcomes across different cell types and genomic loci. Attempts to chemically modify pegRNAs for increased stability can sometimes interfere with their function or fail to provide sufficient protection. Moreover, the lack of robust methods to shield pegRNAs from degradation restricts the scalability and therapeutic applicability of prime editing, particularly in challenging environments such as primary cells or in vivo settings. As a result, there is a pressing need for strategies that can enhance pegRNA stability and, consequently, improve the overall performance and reliability of prime editing systems.

Technology

This genome editing technology enhances the efficiency and precision of prime editing by stabilizing the prime editing guide RNA (pegRNA) component. It achieves this by integrating a small RNA binding exonuclease protection factor La (SSB) protein, or its N-terminal domain, which specifically binds to the 3’-polyuridine tail of the pegRNA. This binding shields the critical 3’ extension—containing the primer binding site and DNA synthesis template—from degradation by exonucleases. The SSB protein can be used as a standalone stabilizing agent or fused directly to the Cas9 nickase-reverse transcriptase complex, forming a single recombinant protein. Additionally, the pegRNA can be chemically modified (e.g., with 2’-O-methylation or phosphorothioate bonds) to further enhance SSB binding and metabolic stability. This system can be delivered as separate or fused components via various vectors and is applicable to a wide range of cell types and genome editing strategies, including substitutions, insertions, deletions, and larger genomic modifications.

What differentiates this technology is its targeted approach to overcoming a key limitation in prime editing: the instability of the pegRNA’s 3’ extension. By leveraging the SSB protein’s high-affinity binding to the polyuridine tail, the system effectively prevents exonuclease-mediated degradation, which has been experimentally shown to dramatically increase editing efficiency—up to 34-fold in some cases. The modularity of the system allows for flexible assembly, including the use of optimized chemical modifications and fusion proteins, making it adaptable for diverse genome engineering applications. Furthermore, the technology is validated across multiple cell types and genomic loci, and is compatible with advanced editing strategies such as PRIME-Del and PASTE. This comprehensive approach—combining biochemical stabilization, genetic engineering, and delivery optimization—sets it apart as a robust and versatile platform for both research and therapeutic genome editing.

Benefits

• Enhanced stability of prime editing guide RNAs (pegRNAs) by protecting their 3’ extensions from exonuclease degradation.
• Significantly increased prime editing efficiency across various genomic loci, cell types, and edit types.
• Modular design allowing fusion of the SSB protein to Cas9 nickase and reverse transcriptase for a single recombinant polypeptide.
• Compatibility with chemical modifications on pegRNAs to optimize binding affinity and metabolic stability.
• Enables precise genetic modifications including substitutions, insertions, deletions, and large-scale genomic edits.
• Applicable to therapeutic genome editing, including treatment of genetic disorders like sickle cell anemia.
• Versatile delivery options including plasmids, RNA, viral vectors, and exosomes for diverse research and clinical applications.

Applications

• Gene therapy for genetic diseases
• Cell line engineering for research
• Generation of animal disease models
• Ex vivo editing of immune cells
• Pharmaceutical genome editing platforms

Additional info

This genome editing system utilizes the N-terminal domain of the small RNA binding exonuclease protection factor La (SSB) protein to stabilize prime editing guide RNAs (pegRNAs). By binding to 3’-polyuridine tails, optionally chemically modified, SSB shields pegRNA extensions from degradation. This stabilization, facilitated by SSB as a standalone component or fused to a Cas9 nickase and reverse transcriptase complex, enhances the efficiency of precise genetic modifications, including substitutions and insertions.