Prime editing, a novel CRISPR-based genome editing tool, eliminates the need for double-stranded DNA breaks or exogenous donor DNA templates, demonstrating great potential in biomedical research and gene therapy. Despite its versatility and precision, Prime editing efficiency varies significantly across different editing types, target sites, and cell types. Thus, improving its efficiency is crucial for broader application.

The molecular mechanism by which the Prime editor recognizes pegRNA and interacts with target DNA remains unclear, limiting the understanding and further optimization of the Prime editing process. To address this issue, researchers determined the cryo-electron microscopy (cryo-EM) structures of the Prime editor in various states, providing a structural framework for understanding this innovative genome engineering system.

Using cryo-EM technology, the researchers analyzed the structures of the Prime editor complex in different states, including pre-initiation, initiation, elongation, and termination. They successfully obtained high-resolution structures of the Prime editor complex in multiple states, revealing its dynamic changes during the guided reverse transcription process. Based on structural information, the researchers designed pegRNA variants and Prime editor variants. They also truncated and fused M-MLV RT, successfully developing a smaller Prime editor variant (PECO-Mini). This variant maintains editing efficiency while improving AAV vector titers and Prime editing efficiency. Activity tests showed that the engineered Prime editor variant has comparable activity to the original Prime editor in vitro. This study reveals the structural characteristics of the Prime editor complex in different working states, providing important information for understanding its molecular mechanism.

Original Article Link: < https://doi.org/10.1038/s41586-024-07259-6 >

While many studies focus on improving the system performance of the Prime editor to enhance its editing efficiency, there is still limited understanding of how Prime editing works within the cellular environment and how its interaction with the cellular context affects editing results. Researchers have attempted to improve Prime editor efficiency by identifying other cellular determinants that influence it and conducting related studies.

Through genome-scale CRISPR interference (CRISPRi) screening , researchers discovered a key Prime editing promoter - the small RNA-binding exonuclease protection factor La. Studies have shown that La protein binds to the 3' polyU sequence of pegRNAs through its N-terminal domain, thereby promoting Prime editing. This effect of La protein is effective for different types of editing (substitution, insertion, deletion) and different cell types. Based on this discovery, the researchers developed a new Prime editor protein (PE7), which fuses the La's RNA-binding N-terminal domain to the PEmax editor. The PE7 editor significantly improves Prime editing efficiency when using expressed pegRNAs, engineered pegRNAs (epegRNAs), and synthetic pegRNAs optimized for La binding.