OR01

POLYPRIME: A novel DNA-Polymerase based prime editing strategy

J R Alvarez Vargas(1,2) A De Cian(1) A Brion(1) K Lamribet(1) F Gourmelon(1) G Gentile(1,2) J P Concordet(1) C Giovannangeli(1)

1:Museum National d'Histoire Naturelle ; Inserm U1154 ; CNRS UMR7196; 2:Université Paris Saclay

Prime editing (PE) is a DSB-free method that edits DNA using a Cas9 nickase fused with reverse transcriptase (RT), and a modified guide RNA (pegRNA) with a 3’ extension. However, PE efficiency depends on the target, cell type, and edit size, with inefficient long insertions or replacements (>50bp). In addition, the scaffold sequence from the pegRNA can be incorporated into the genome.

Our goal is to develop safer and more efficient PE tools. We created a DNA Polymerase-based method called POLYPRIME. We replaced RT with DNA polymerases (DNApols), which could help to install long edits and reduce scaffold incorporation. POLYPRIME uses a hybrid RNA-DNA molecule, pegDNA, which we produce by ligating a standard gRNA to a DNA extension.

We evaluated POLYPRIME in vitro, comparing three DNApols. Our results showed that pegDNAs can generate target nicks more efficiently than standard pegRNAs, and the 3’ DNA extension is efficiently copied by Cas9-DNApol fusions. Next, we optimized POLYPRIME in cells using a reporter system based on BFP to GFP conversion (due to C to T conversion). Different lengths and chemistries of the 3’ DNA extension were tested to optimize the pegDNA design, finding that the 3’extension should be much longer in pegDNAs compared to pegRNAs, typically 20nt compared to 10. We tested 3 DNA-pol fusions with optimized pegDNAs, all showing equivalent POLYPRIME activity. We validated the optimized POLYPRIME design on endogenous targets and found equivalent or superior activity to standard PE, especially with long insertions (30 to 54 bp).