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B - Gene Targeting and Gene Correction -> B2 - Gene Targeting and Gene Correction – In Vitro Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

523: Reducing the Inherent Auto-Inhibitory Interaction within the pegRNA Enhances Prime Editing Efficiency

Type: Poster Session

Poster Board Number: 523
Presentation Details
Session Title: Wednesday Poster Session
Start Time: 5/17/2023 12:00
End Time: 5/17/2023 14:00

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 local sequence composition. However, characterization of optimal PBS length has been based on prime editing outcomes using plasmid or lentiviral expression systems. Here we report that the inherent complementarity between the PBS and spacer sequence within the pegRNA affects pegRNA binding efficiency and target recognition. Because RNA-RNA duplexes are typically more stable than RNA-DNA duplexes and due to the intramolecular nature of the association between the spacer and PBS regions of the pegRNA, the formation of a PBS-spacer RNA duplex can preclude the formation of an R-loop by the prime editor at its target site. Finding the correct balance between PBS length and pegRNA sequence composition is critical to maximize prime editing activity by reducing the inherent “auto-inhibition” within the pegRNA sequence composition. We demonstrate that reducing the complementarity between the PBS and spacer sequences by reducing PBS length can broadly improve prime editing activity in the context of prime editor protein complexed with synthetic, end-protected pegRNAs across multiple target sites and multiple cell types. The difference in PBS length requirement between plasmid expression systems and PE RNPs loaded with synthetic, end-protected pegRNAs appears to originate from the susceptibility of transcriptionally produced pegRNAs to 3’ end degradation. By small RNA sequencing, we observe that while the majority of the transcriptionally produced pegRNAs are full length, the pegRNA species that are bound by the prime editor protein are enriched for 3’ truncated species. In the case of end-protected pegRNAs, we demonstrate that shorter PBS lengths with a PBS-target strand melting temperature near 37°C are optimal. Additionally, a transient cold shock treatment of the cells post PE-pegRNA delivery further increases prime editing outcomes. 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 install precise edits in primary human T cells and zebrafish.

Karthikeyan Ponnienselvan, Pengpeng Liu, Thomas Nyalile, Sarah Oikemus, Stacy A. Maitland, Nathan D. Lawson, Jeremy Luban, Scot A. Wolfe

UMass Chan Medical School, Worcester, MA
 K. Ponnienselvan: None.

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