Analyses of OT effects. (A) OT editing measured using amplicon deep sequencing at the top 20 potential CIRCLE-seq nominated sites. The workflow is illustrated on the left side. Genomic DNA was isolated from BM MNCs of an untreated mouse or the mouse showing highest on-target editing after in vivo transduction with HDAd-PE5max. OT prime editing (middle bar graph) was calculated based on the percentage of reads with the G/T/C > A conversion at position +4 (corresponding to sickle mutation), counting the predicted nicking site as position +1. If position +4 was already an A in the wild-type allele, the calculation was performed based on the percentage of G/T/C > A conversion at position +5 (corresponding to the silent PAM mutation). Percentage of reads with indels was also analyzed (right bar graph). (B) OT editing at the top-5 ranked potential sites in mouse genome nominated through in silico prediction using Cas-OFFinder. Genomic DNA samples were the same as those in A. The workflow is illustrated on the left side. (C) OT editing at the top-5 ranked potential sites in human genome nominated through in silico prediction using Cas-OFFinder. Genomic DNA was isolated from untreated or HDAd-PEmax–transduced and selected SCD CD34⁺ cells. Percentage of reads with prime editing and indels are shown. Note that hOT#1’ (highlighted by a $ symbol) maps to the HBD site and only bears 1 bp mismatch at the on-target site.