Abstract
The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.
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Data availability
Tracking-seq data have been deposited in the National Center for Biotechnology Informationʼs Gene Expression Omnibus with accession number GSE236360 (ref. 51). Source data are provided with this paper. All other data are available in the Supplementary Information.
Code availability
Code for Tracking-seq analysis is available on GitHub (https://github.com/Lan-lab/offtracker)52.
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Acknowledgements
We thank H. Ren and T. Wei (Institute of Zoology, Chinese Academy of Sciences) for their help with mouse liver delivery. We thank W. Shao and H. Qi (School of Medicine, Tsinghua University) for providing mouse B cells. This work was partially supported by grants from the National Natural Science Foundation of China (grant no. 81972680 to X. Lan), the Beijing Natural Science Foundation (grant no. Z210010 to Y.L.), the Tsinghua University-Peking University Joint Center for Life Science (grant no. 61020100119 to X. Lan), the Beijing Natural Science Foundation (grant no. 20201100463 to X. Lan), the Tsinghua University Initiative Scientific Research Program (grant no. 2022Z11QYJ032 and grant no. 2021Z11JCQ020 to Y.L.), the National Natural Science Foundation of China (grant no. 32171448 to Y.L.) and the National Key R&D Program of China (grant no. 2019YFA0904402 and grant no. 2019YFA0906700 to Y.L.) M.Z. is supported by a Tsinghua-Peking Center for Life Sciences postdoctoral fellowship.
Author information
Authors and Affiliations
Contributions
M.Z., Y.L. and X. Lan designed the study and experiments. M.Z. and J.W. developed the Tracking-seq experimental protocol. M.Z., J.W. and Y.Y. conducted Tracking-seq experiments. R.X. developed the algorithm for analyzing Tracking-seq data and analyzed all sequencing data. J.Y. and M.Z. conducted genome editing on cell lines. T.C. conducted genome editing on mHSPCs. J.Y., M.Z. and A.J. conducted genome editing on mouse liver. R.X., X.R., J.Y. and H.T. conducted targeted amplicon sequencing. H.W., P.Z., J.D. and X. Lin helped with the delivery into mouse livers. H.Y. and C.L. participated in discussions. X. Lan, Y.L. and W.X. supervised the experiments. M.Z., R.X., Y.L. and X. Lan wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
Tsinghua University has submitted a patent application (PCT/CN2023/071055) on the method for off-target detection used in this study, with M.Z., Y.L. and X. Lan as inventors. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Highly distinguishable signals produced by Tracking-seq.
a, The DNA cleavage sites were located at the center of symmetry of the Tracking-seq signal. b, Boxplot showing the signal lengths of different editing tools at different time points after transfection with HEK293_site_4 (n = 1 for each condition). Each dot indicates an editing site. Top 10 sites were shown for Cas9, ABE, and CBE; All 5 positive sites were shown for PE2. Boxplot format: center line: median; box limits: quartiles; whiskers: 1.5 × interquartile range (IQR). c, Boxplot showing the signal intensity bias of different editing tools for HEK293_site_4 and VEGFA_site_2 (n = 1 for each condition). Cas9 signals showed no bias while BEs and PEs signals tended to enrich in non-nicked strands. The non-nicked strands of BEs are the PAM-containing strands while those of PEs are the opposite of PAM-containing strands. Each dot indicates an editing site. P-values were calculated by the one-sided Wilcoxon rank-sum test. Boxplot format: center line: median; box limits: quartiles; whiskers: 1.5 × interquartile range (IQR). d, The schematic diagram illustrates a quantitative method for determining the relative proportion of single-stranded DNA (ssDNA) upstream and downstream of the editing site. Two restriction enzyme sites equidistant from the cleavage site of the editing site were selected. qPCR primers were designed near the enzyme cutting sites to allow for binding of upstream and downstream primers on either side of the cleavage site. Genomic DNA from editing cells was digested using the corresponding restriction enzyme. Double-stranded DNA containing the enzyme cutting sites near the editing site was cleaved, while ssDNA remained intact for qPCR amplification. By normalizing the qPCR cycle threshold (Cq) values to those of the corresponding genomic positions in wild-type (WT) cells, the relative quantities of ssDNA upstream and downstream of the editing site were quantified. e, Boxplot showing the relative quantities of ssDNA 1 kb upstream and downstream of the on-target site of HEK293_site_4. ABE and PE2 showed more ssDNA in the upstream regions, which suggested ssDNA was enriched in the PAM-containing strand for both ABE and PE2 at the on-target site. Three technical replicates were performed for each condition. Error bars indicate ± SD. f, Correlation of off-target Track scores in experiments using 200,000 cells (left) or 5,000 cells (right) compared to those obtained with 500,000 cells. Each dot represents an off-target site. g, Tracking-seq signals at two off-target sites with different initial numbers of pure GFP+ cells (dark blue tracks) or cells mixing varying numbers of GFP+ cells with wild-type (WT) cells up to a total of 100,000 cells through a gradient dilution (light blue tracks). h, Track scores and corresponding indel rates obtained from different proportions of GFP+ cells through the gradient dilution (100,000 cells in total). Blue bars and the left y-axis indicate the Track score; red dots, percentage numbers, and the right y-axis indicate the indel rate; the dashed line indicates the threshold of the Track score for positive sites.
Extended Data Fig. 2 Dynamics of Tracking-seq signal over time for HEK293T with sgRNA HEK293_site_4.
a, The signal of the on-target site and an off-target site were shown. The condition is 500, 000 HEK293T cells with sgRNA HEK293_site_4. b, The dynamics of Tracking-seq signals over time (n = 1 for each condition). The time points indicate the periods after the PEI transfection of HEK293T cells. Background indicates the mean score of all negative sites (see Methods for more details). c, The expression kinetics of editing proteins (left) and guide RNAs (right) measured by the fluorescent intensity at different time points after transfrection. (n = 1 for each condition).
Extended Data Fig. 3 Comparison of the number of editing sites detected by Tracking-seq and GUIDE-seq.
a, Venn diagrams comparing Cas9-induced editing sites detected by Tracking-seq with those detected by GUIDE-seq for 9 different sgRNAs. b, The number of detected editing sites of Tracking-seq and GUIDE-seq were highly correlated. Each dot indicates a sgRNA. ρ indicates Spearman’s rank correlation coefficients.
Extended Data Fig. 4 Performance evaluation of four off-target detection methods with HEK293T cells edited by Cas9 and HEK293_site_4 or VEGFA_site_2.
a, The numbers of detected off-target sites from HEK293T cells edited with Cas9 and HEK293_site_4 (left) or VEGFA_site_2 (right) in this study were comparable to those detected by Zou et al. Nat. Methods 2023. b, Boxplot showing the distribution of Track scores, DISCOVER scores, DISCOVER+ scores, and GUIDE scores among different indel ranges for HEK293_site_4 and VEGFA_site_2 (n = 1 for each condition). Each dot indicates a site tested by amplicon sequencing. Fractional numbers in blue indicate true-positive proportion and those in red indicate false-positive proportion. c, Comparisons of Track scores and scores from three other methods for HEK293_site_4 and VEGFA_site_2. Each dot indicates a site from the pool of collected candidate off-target sites. ρ indicates Spearman’s rank correlation coefficients.
Extended Data Fig. 5 Genome tracks of representative off-target sites.
a-b, Signals generated by Tracking-seq, DISCOVER-seq, and DISCOVER-seq+ at representative off-target sites for HEK293_site_4 (a) and VEGFA_site_2 (b). ‘+’ or ‘-’ under the ‘Detection’ columns indicates whether the site was detected or not by the corresponding method. The indel rates shown below the tracks were measured by amplicon sequencing. c, Signals at off-target sites with indel <0.01% but showing the distinct Tracking-seq pattern for HEK293_site_4 (left) and VEGFA_site_2 (right). Rep1 and rep2 were two biological replicates. WT HEK293T cells were used as control.
Extended Data Fig. 6 CBE, CBE-dCas9, and PE2 edited with HEK293_site_4.
a-b, Bar chart shows the C-to-T rates of all tested sites detected by Tracking-seq (a) and not detected by Tracking-seq (b). Cytosines upstream of the protospacer, cytosines within the protospacer, indels, and background cytosines are in blue, pink, purple, and grey, respectively. Background refers to cytosines located outside the protospacer but within ±50 bp from the 5′ end of the protospacer. Two technical replicates were performed for each site. Bars with one point and no error bar indicates PCR failures for one of the two replicates. Error bars indicate ± SD. c, The editing efficiency of CBE with dCas9 at the on-target site of HEK293_site_4, showing ~23% C to T conversion in the editing window. d, Tracking-seq signals at the on-target site. e, Comparison of the Track scores of CBE with nCas9 and CBE with dCas9. Each dot represents a candidate off-target site; the dashed line indicates the threshold for positive sites. f, Tracking-seq signals at a representative off-target site. g, Bar charts showing editing rates of off-target sites induced by PE2 detected by Tracking-seq. Dots in the bar charts indicate biological replicates. Precise editing refers to sequence changes that exactly match the reverse transcriptase template at the on-target site. All the other mutations fall into ‘undesirable substitution’ or ‘undesirable indel’. h, Sequences related to Off-2 with unusually high indel rates in g. The last four rows indicate representative sequences detected in the amplicon sequencing.
Extended Data Fig. 7 Track-seq signals around on-target and off-target sites of PE editing.
Samples were HEK293T cells edited by PE2-HEK293_site_4, PE2-VEGFA_site_2, or PE3-VEGFA_site_2.
Extended Data Fig. 9 Comparison of track scores in different conditions.
a, Consistency of Tracking-seq between replicates. Batch1 and batch2 were two biological replicates while batch2_1 and batch2_2 were two technical replicates. The condition is 500, 000 HEK293T cells with sgRNA HEK293_site_4 after 96-hour Cas9 editing. Each dot indicates a candidate site. b, Comparison of track scores between PE2 and PE3 using VEGFA_site_2 in HEK293T cells. c, Comparison of track scores between 150, 000 cells and 60, 000 cells of mHSPCs with Cas9 editing using Pcsk9-gP+G. d, Comparison of track scores among mHSPCs, mESCs, NIH3T3 cells, and AML12 cells with Cas9 editing using Pcsk9-gP+G. For mHSPCs, the mean track score of the two experiments (150, 000 cells and 60, 000 cells) was used. For mESCs, one experiment of 30,000 cells was conducted. For NIH3T3, the mean track score of the two experiments (100, 000 cells and 200, 000 cells) was used. For AML12, one experiment of 100,000 cells was conducted. The orange dots and red dots represent the tested differential off-target sites (the same as Fig. 6a).
Extended Data Fig. 10 Heterogeneity of off-target sites.
a, Scatter plots showing the correlation between Track scores and indel rates of 4 cell types at 7 differential off-target sites. b, The wild-type genome sequences of NIH3T3 cells, AML12 cells, mHSPCs, and mESCs around tested off-target sites. Dots in the bar charts indicate biological replicates. c, Indel rates and Track scores of the off-target sites Dif-04 to Dif-07 in four cell types and their ATAC-seq, H3K9me3 ChIP-seq, and H3K27ac ChIP-seq signals. Dots in the bar charts indicate biological replicates. d, Enrichment of epigenetic profiles around off-target sites of Pcsk9-gP+G in NIH3T3 cells, AML12 cells, mHSPCs, and mESCs.
Supplementary information
Supplementary Information
Supplementary Figs. 1–10, Tables 1–4 and methods.
Supplementary Table 5
Sequences of sgRNAs and pegRNAs.
Supplementary Table 6
Comprehensive results of four methods.
Supplementary Table 7
Other Tracking-seq and amplicon results for human.
Supplementary Table 8
Tracking-seq and amplicon results for mouse.
Supplementary Table 9
All primer sequences.
Source data
Source Data Figs. 1, 2, 5 and 6 and Extended Data Figs. 1–3, 6 and 9
Statistical source data.
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Zhu, M., Xu, R., Yuan, J. et al. Tracking-seq reveals the heterogeneity of off-target effects in CRISPR–Cas9-mediated genome editing. Nat Biotechnol 43, 799–810 (2025). https://doi.org/10.1038/s41587-024-02307-y
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DOI: https://doi.org/10.1038/s41587-024-02307-y
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