PhD Scientific Days 2026

Poster Session 1.A - Molecular Medicine

Disrupting pegRNA intramolecular complementarity via primer binding site and spacer sequence alterations can enhance prime editing efficiency

Name of the presenter

Biczók, Zsuzsanna

Institute/workplace of the presenter

School of Ph.D. Studies, Semmelweis University

Authors

Zsuzsanna Biczók^1,2,3, Sarah Laura Krausz¹, Dorottya Anna Simon^1,2,4, Eszter Tóth¹, Éva Varga^1,5, Tamás Annus⁶, Flóra Huba⁶, Máté Varga⁶, Éva Bakos¹, Elfrieda Fodor⁷, Ervin Welker^7,8
1: Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences
2: School of Ph.D. Studies, Semmelweis University
3: Gene Design Kutató Fejlesztő Kft.
4: Hun-Gén Technologies Kft.
5: School of PhD Studies, University of Szeged
6: Deparment of Genetics, ELTE Eötvös Loránd University
7: Institute of Biochemistry, HUN-REN Biological Research Centre
8: Institute of Molecular Life Sciences, HUN-REN Research Centre for Natural Sciences, Budapest, Hungary

Text of the abstract

Introduction
Prime editing is a highly versatile genome editing method, but its efficiency is limited by multiple bottlenecks inherent to its complex mechanism, requiring extensive optimization. A key constraint is the intramolecular complementarity between the pegRNA spacer and the primer binding site (PBS), whose length and sequence are critical yet can negatively impact editing outcomes. To investigate this, we analyzed over 300 modified constructs by introducing mismatches in the spacer or PBS, as well as small deletions within the PBS sequence.
Aims
We investigated how reducing pegRNA intramolecular complementarity (via spacer/PBS mismatches and varying PBS lengths) affects prime editing efficiency.
We analyzed how 1–4 nt deletions in a 20 nt PBS, alone and combined with spacer mismatches, influence efficiency and assessed the role of the remaining PBS tail.
We determined the impact of a single-nucleotide PBS deletion across multiple target genes and two cell lines, including its effect on off-target activity.
Methods
Molecular cloning; HEK293T and hPSC cell culturing, transfection and nucleofection; Genomic DNA purification and PCR; Next generation sequencing; In vitro RNA transcription; Protein purification; Data analysis and statistics
Results
We found that reducing spacer–PBS complementarity—via mismatches, small deletions in a long (20 nt) PBS, or their combination—can enhance prime editing efficiency, sometimes surpassing optimized pegRNAs. Notably, a single-nucleotide deletion at position 13 of the PBS achieved efficiencies comparable to short PBS designs without increasing off-target activity. We termed this simplified approach SPELL (Streamlined Prime Editing with fixed-Length PBS Leverage).
Conclusion
In conclusion, reducing spacer–PBS complementarity seemingly inhibits prime editing, and its alleviation via the SPELL strategy enhances efficiency, often reaching optimized designs and enabling high efficiency in most tested edits without extensive optimization
Funding
The project was supported by grants K134968, K142322 from the Hungarian Scientific Research Fund (OTKA); by “PharmaLab” (RRF-2.3.1-21-2022-00015) from the National Research, Development, and Innovation Office of Hungary; by ELKH-PoC-2023 form the Hungarian Research Network (HUN-REN) and by GINOP_PLUSZ-2.1.1-21-2022-00049 to GENE DESIGN Kft. from the Ministry of National Economy.