A flaw that impeded the 'magic' of prime editing made 7‑fold more powerful by design optimization
1. Hidden Pitfall of Precise Gene Correction: Intramolecular Complementarity
Prime Editing is a technology that can correct genes with extreme precision, but an unexpected trap emerged during the design process. The guide RNA (pegRNA) that facilitates editing can fold onto itself due to "intramolecular complementarity," frequently causing a sharp drop in editing efficiency.
2. SPELL Strategy: Breaking the Conventional Notion of Complex Design
To address this issue, the research team tested more than 300 variant RNAs and developed a new design method called SPELL (Streamlined Prime Editing with fixed‑Length PBS Leverage). Rather than relying on complex calculations, the approach fine‑tunes the length of the primer binding site (PBS) and intentionally introduces a small deletion at a specific position, preventing RNA misfolding and ensuring precise attachment to the target site.
3. Results: Up to Seven‑fold Efficiency Gain Without Prior Optimization
The experimental results were striking. Whereas previously many rounds of trial‑and‑error were required to boost efficiency, applying the SPELL method caused editing efficiencies to rise up to seven‑fold for most targets without any special pre‑optimization. This provides a powerful tool for standardizing the otherwise complex gene‑editing workflow.
4. Future Significance and Outlook
If the SPELL technology becomes widely adopted, the barrier to gene editing will be dramatically lowered. As editing becomes easier and more accurate for all users, the development of personalized therapeutics for rare genetic diseases and functional genomics research are expected to progress at a much faster pace than before.
The length and sequence of the primer binding site (PBS) are critical for efficient prime editing, and its intramolecular complementarity with the prime editing guide RNA (pegRNA) spacer is a major drawback. We investigated the effects of these factors by literature analyses and by testing over 300 modified pegRNAs with weakened PBS-spacer interactions. It has been suggested that the effective PBS length for plasmid-delivered pegRNAs without end protection is considerably longer than what efficient priming requires due to exonuclease digestion of the PBS ends; however, analysing literature data of over 3000 pegRNAs revealed no significant shift in the optimal PBS length for epegRNAs compared to conventional pegRNAs. We also found improvement in editing efficiency with up to seven-fold when mismatches were introduced in the spacer or PBS sequence disrupting complementarity, although this effect is more pronounced with non-optimal PBS lengths. A combination of spacer mismatches and PBS deletions led to further editing improvements, even compared to the optimal PBS, although finding the best combination requires extensive optimization. Here, we achieved near-optimal editing efficiency in the majority of cases without the need for prior pegRNA optimization by using SPELL ("Streamlined Prime Editing with fixed-Length PBS Leverage"), a prime editing approach that employs a 17-20 nucleotide-long PBS with a single nucleotide deletion.
It overcomes the technical hurdle that overly complex and low‑efficiency gene‑editing designs have posed for clinical application. By simplifying and improving the accuracy of the correction process, patients with genetic disorders can receive safe, genotype‑matched therapies more rapidly.