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editing is a versatile tool for precise genome engineering. A prime editor (PE), typically a Cas9 nickase and a reverse transcriptase (RT) fusion, installs various small edits by copying a template encoded directly by a prime editing guide RNA (pegRNA). RT creates a 3-prime genomic flap which is subsequently incorporated into the target locus by annealing and ligation to form the final prime edit. However, reverse transcription can continue past the intended template, leading to the incorporation of the pegRNA scaffold sequence into the newly formed genomic 3-prime flap. During the integration of the 3-prime flap into the genome, these scaffold-derived sequences can be installed alongside the intended edit into the target locus, thus reducing the precision of prime editing. Here, we develop a method that precisely blocks RT before it reaches the scaffold, to prevent these undesired editing events. We demonstrate that synthetic pegRNAs, modified with an abasic spacer or 2-O-methylation, can precisely and robustly block RT directly at the end of the RT template. This eliminates undesired scaffold-derived sequences from the 3-prime flap and their subsequent incorporation into the target locus."],"repository":["ENA"],"additional_accession":[]},"is_claimable":false,"name":"","description":"Modified pegRNAs mitigate scaffold-derived prime editing by-products","dates":{"last_updated":"2025-02-22","first_public":"2025-02-22"},"accession":"PRJNA1226148","cross_references":{}}