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. 2023 Feb 16;41(10):1446–1456. doi: 10.1038/s41587-023-01678-y

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© The Author(s) 2023

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. 3 — a, Schematic of molecular steps involved in prime editing. b, Normalized pegRNA count derived from sequencing of PCR amplicons from genomic DNA (x axis) or PCR amplicons from RNA (y axis) for the HEK3 site in HEK293T cells for individual pegRNAs (markers). Pink, inserts with four or more consecutive adenines. Data represent the average of n = 3 biological replicates. c, Top panel, average insertion rate relative to length bin median (y axis) for inserts stratified by the longest consecutive run of adenines (x axis). Bottom panel, instead showing transcription rate (read counts from RNA/read counts from DNA) on the y axis. Data are presented as mean ± s.e.m. n = 3 biological replicates. d, Insertion frequencies at the HEK3 site in HEK293T using the standard MMLV reverse transcriptase (PE2, x axis) and the FeLV reverse transcriptase (PE-FeLV, y axis) for different insertion sequences (markers). Colors, number of neighboring points. n = 3 biological replicates. e, As d, but comparing PE3 and PE2 at the EMX1 site. f, Schematic of screens with overexpression constructs. g, Insertion frequencies for different overexpressions (y axis and panels) compared with no overexpression (x axis) for three biological replicate screens (markers) stratified by insertion sequence lengths (colors). h, Average insertion rates (y axis) across insert lengths (x axis) with at least 30 measured sequences for overexpression constructs (colors). Data are presented as mean ± s.e.m. n = 3 biological replicates. i, As h, but instead displaying the insertion rate fold changes of screens with overexpressions compared with no overexpression (y axis), calculated from the ratio of sums of all sequences (lines) or of ten randomly sampled sequences. j, Top, average insertion frequency (grayscale) of four sequences with varying lengths (x axis) when overexpressing eGFP stratified by homology arm lengths (panels). Bottom, insertion rate fold changes compared with eGFP (y axis) when overexpressing TREX1 and TREX2 (colors). n = 2 biological replicates. k, Fraction of the nontemplated adenine allele at the +9 position (y axis) for cells with overexpression constructs (x axis and colors) stratified by experiment and homology arm lengths (panels). Markers show screen averages from three biological replicates for the pooled screen or from separate pegRNAs for the individual validation experiment.