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. 2022 Jun 27;13:3685. doi: 10.1038/s41467-022-31322-3

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — a Example diagram of the PE systems editing MYC + 5GtoC. While nCas9 (H840A) and pegRNA are required for all prime editing strategies, PE3 contains a nicking sgRNA at +42 bp from the pegRNA-mediated nick to increase the editing efficiency. PE3b contains a sgRNA (−5 bp from the pegRNA-mediated nick) with spacer that match the edited strand to minimize the presence of concurrent nicks. Nick only for PE3 introduces a single-strand break in the non-edited strand by a nicking sgRNA. b Numbers of L1-ORFeus insertions obtained by amplicon sequencing at the MYC locus targeted by PE2 (MYC + 2-4AAA del), PE3 (MYC + 2-4AAA del), or nick only for PE3 in HEK293T cells expressing L1-ORFeus, L1-ENm, L1-RTm, or GFP control. Number of L1-ORFeus insertions was pooled from three independent experiments with similar results. c Numbers of L1-ORFeus insertions obtained by amplicon sequencing at the MYC locus targeted by PE2 (MYC + 5GtoC), PE3 (MYC + 5GtoC), PE3b (MYC + 5GtoC), or nick only for PE3/PE3b in HEK293T cells expressing L1-ORFeus or L1-RTm. d Numbers and fragment lengths of L1-ORFeus insertions obtained by amplicon sequencing at the MYC locus targeted by PE3 (MYC + 2-4AAA del) in HEK293T cells expressing L1-ORFeus or L1-RTm. Example L1-ORFeus insertional sequences in cells expressing L1-ORFeus are shown in Supplementary Sequence 4. Example L1-ORFeus insertions bridging the intron of GFP in cells expressing L1-ORFeus are shown in Supplementary Sequence 5. e Pie charts show the relative abundance of four main types of insertions obtained by amplicon sequencing at the MYC locus targeted by PE3 (MYC + 2-4AAA del) in HEK293T cells expressing L1-ORFeus, L1-ENm, L1-RTm, or GFP control. f Detailed view of the distribution of L1-ORFeus insertions at the MYC locus obtained by PolyA-seq in HEK293T cells targeted by PE3 (MYC + 2-4AAA del), PE3 (MYC + 5GtoC), or PE3b (MYC + 5GtoC) that express L1-ORFeus or L1-RTm.