PUBLICATION
Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency
- Authors
- Ponnienselvan, K., Liu, P., Nyalile, T., Oikemus, S., Maitland, S.A., Lawson, N.D., Luban, J., Wolfe, S.A.
- ID
- ZDB-PUB-230530-29
- Date
- 2023
- Source
- Nucleic acids research 51(13): 6966-6980 (Journal)
- Registered Authors
- Lawson, Nathan, Wolfe, Scot A.
- Keywords
- none
- MeSH Terms
-
- Animals
- Binding Sites
- CRISPR-Cas Systems
- Cold Temperature*
- Cold-Shock Response
- Gene Editing*
- Humans
- Mammals
- Ribonucleoproteins
- Zebrafish*/genetics
- PubMed
- 37246708 Full text @ Nucleic Acids Res.
Citation
Ponnienselvan, K., Liu, P., Nyalile, T., Oikemus, S., Maitland, S.A., Lawson, N.D., Luban, J., Wolfe, S.A. (2023) Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency. Nucleic acids research. 51(13):6966-6980.
Abstract
Prime editing systems have enabled the incorporation of precise edits within a genome without introducing double strand breaks. Previous studies defined an optimal primer binding site (PBS) length for the pegRNA of ∼13 nucleotides depending on the sequence composition. However, optimal PBS length characterization has been based on prime editing outcomes using plasmid or lentiviral expression systems. In this study, we demonstrate that for prime editor (PE) ribonucleoprotein complexes, the auto-inhibitory interaction between the PBS and the spacer sequence affects pegRNA binding efficiency and target recognition. Destabilizing this auto-inhibitory interaction by reducing the complementarity between the PBS-spacer region enhances prime editing efficiency in multiple prime editing formats. In the case of end-protected pegRNAs, a shorter PBS length with a PBS-target strand melting temperature near 37°C is optimal in mammalian cells. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes for pegRNAs with optimized PBS lengths. Finally, we show that prime editor ribonucleoprotein complexes programmed with pegRNAs designed using these refined parameters efficiently correct disease-related genetic mutations in patient-derived fibroblasts and efficiently install precise edits in primary human T cells and zebrafish.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping