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. 2022 Jan 25;13:474. doi: 10.1038/s41467-022-28028-x

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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. 1 — a, b A schematic representation of paired gRNA-target design using a single-target or b dual-target sequence. c The design of control dual-target sequences corresponding to 4 cleavage types, where the left and right protospacers are identical and the cleavage events are turned on or off by “NGG” or “NTT” PAM sequences, respectively. d Bar charts showing the fractions of five types of editing outcomes in relation to the 4 cleavage types. Left: a gRNA associated with dominating non-homologous end joining (NHEJ); right: a gRNA associated with dominating microhomology-mediated end joining (MMEJ), where the microhomology sequences are highlighted in red rectangles. e A demonstration of the cleavage-editing model of the dual-target system. f A heatmap showing the cleavage-editing transition matrix computed from mutational profiles of 276 control dual-target sequences. g Scatter plots showing the correlations of off-on ratios between biological replicates using single- or dual-target systems. h A box plot showing the distributions of the off-on ratios estimated by the dual-target system, corresponding to the 32 filtered benchmark gRNAs and 480 target sequences. Positive: reported off-target sequences; Negative: unreported genomic sequences with <5 mismatches relative to crRNA. The data represent n = 204 positive, 213 negative (in vitro), and n = 66 positive, 52 negative (in vivo) gRNA-target pairs. The box plot displays a median line, interquartile range boxes and min to max whiskers. The p-values were calculated using two-tailed Manny–Whitney U test. i, j Scatter plots showing the correlations between the off-on ratios estimated from the dual-target system and i GUIDE-seq or j WGS, at the reported genomic off-target sequences. The p-values were calculated using Pearson correlation test. The shadow represents the 95% confidence interval. k A demonstration of the off-target sequence design in the high-throughput experiments using the dual-target synthetic system, where the mismatches in the 2-MM and 3-MM sequences are the combinations of the mismatches in the 1-MM sequences. Source data for Fig. 1d, g–j are provided in the Source Data file.