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Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag

Nature Methods volume 20, pages 898–907 (2023)Cite this article

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Abstract

Prime editors have a broad range of potential research and clinical applications. However, methods to delineate their genome-wide editing activities have generally relied on indirect genome-wide editing assessments or the computational prediction of near-cognate sequences. Here we describe a genome-wide approach for the identification of potential prime editor off-target sites, which we call PE-tag. This method relies on the attachment or insertion of an amplification tag at sites of prime editor activity to allow their identification. PE-tag enables genome-wide profiling of off-target sites in vitro using extracted genomic DNA, in mammalian cell lines and in the adult mouse liver. PE-tag components can be delivered in a variety of formats for off-target site detection. Our studies are consistent with the high specificity previously described for prime editor systems, but we find that off-target editing rates are influenced by prime editing guide RNA design. PE-tag represents an accessible, rapid and sensitive approach for the genome-wide identification of prime editor activity and the evaluation of prime editor safety.

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Fig. 1: DNA tag integration at target site and off-target sites by prime editor in human cells.
Fig. 2: Genome-wide detection of prime editing by PE-tag in vitro using genomic DNA.
Fig. 3: Genome-wide detection of prime editing by PE-tag in cells.
Fig. 4: In vivo PE-tag in mouse liver.
Fig. 5: Identification of off-target prime editing associated with therapeutically relevant targets.

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Data availability

Illumina sequencing data have been submitted to the Sequence Read Archive. mm10 and hg38 were used as reference genome. These datasets are available under BioProject accession number PRJNA811252. The authors declare that all other data supporting the findings of this study are available within the paper and its Supplementary Information files. Backbone plasmids used for pegRNA and sgRNA cloning are available from Addgene. Source data are provided with this paper.

Code availability

The software used for data analysis is available at Github (Supplementary Note 6; https://github.com/umasstr/GS-Preprocess and https://rdrr.io/github/LihuaJulieZhu/GUIDEseq/man/PEtagAnalysis.html).

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Acknowledgements

We thank members of the Xue Lab and Wolfe Lab for helpful discussions. We thank H. Valley and M. Mense at the Cystic Fibrosis Foundation Therapeutic Lab for providing HBE cells. W.X. was supported by grants from the National Institutes of Health (DP2HL137167, P01HL158506 and UH3HL147367) and the Cystic Fibrosis Foundation. S.A.W., P.L. and K.P. were supported in part by the National Institutes of Health (grants R01HL120669 and UG3TR002668) and the Rett Syndrome Research Trust. C.K. and P.C. were funded by the Synthetic Biology Platform at the Wyss Institute for Biologically Inspired Engineering and by the MIT Media Lab consortia of sponsors.

Author information

Author notes

  1. These authors contributed equally: Shun-Qing Liang, Pengpeng Liu.

Authors and Affiliations

  1. RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Shun-Qing Liang, Zexiang Chen, Erik J. Sontheimer & Wen Xue

  2. Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Pengpeng Liu, Karthikeyan Ponnienselvan, Sneha Suresh, Lihua Julie Zhu, Wen Xue & Scot A. Wolfe

  3. Wyss Institute, Harvard Medical School, Boston, MA, USA

    Christian Kramme & Pranam Chatterjee

  4. Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA

    Pranam Chatterjee

  5. Department of Biomedical Engineering, Duke University, Durham, NC, USA

    Pranam Chatterjee

  6. Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Lihua Julie Zhu, Erik J. Sontheimer & Wen Xue

  7. Program in Bioinformatics and Integrative Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Lihua Julie Zhu

  8. Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Erik J. Sontheimer, Wen Xue & Scot A. Wolfe

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Contributions

S.-Q.L. and P.P.L. performed experiments, analyzed data and wrote the manuscript with co-authors. K.P. prepared protein. S.S. generated the HEK293T1278+TATC cell line. Z.C. prepared PE2 mRNA. C.K. and P.C. generated the HEK293TT158M cell line. L.J.Z. performed bioinformatic analysis. E.J.S., W.X. and S.A.W. supervised the study and wrote the manuscript with all co-authors.

Corresponding authors

Correspondence to Pengpeng Liu, Wen Xue or Scot A. Wolfe.

Ethics declarations

Competing interests

University of Massachusetts has filed a patent application (serial no. 63/328076) on PE-tag in this work. S.A.W. is a consultant for Chroma Medicine and serves on the S.A.B. for Graphite Bio. W.X. is a consultant for the Cystic Fibrosis Foundation Therapeutics Lab. All remaining authors declare that the research was conducted in the absence of commercial or financial conflict of interest. The authors declare no competing nonfinancial interests.

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Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editors: Lei Tang and Madhura Mukhopadhyay, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 DNA-tag integration at target site and off target sites by PE2.

a, Comparison of the PE2 prime editing efficiency as a function of different tag and HA lengths within the pegRNA at the Pcsk9 target site and OT-1 in Hepa1-6 cells, where prime editing components were delivered by transient transfection. Insertion rates at the target site are precise edits, whereas percent editing at OT1 captures indels as well as tag insertions. Frequencies of editing were quantified by deep sequencing from PCR amplicons spanning each locus. Results were obtained from three independent experiments and presented as mean ± SD. b, Comparison of the prime editing efficiency for different tag and HA lengths inserted by PE2 at the VEGFA target site and OT-1 in HEK293T cells. Editing rates are determined by Illumina sequencing PCR amplicons spanning each locus. Frequencies of editing were quantified by deep sequencing from PCR amplicons spanning each locus. Results were obtained from three independent experiments and presented as mean ± SD.

Source data

Extended Data Fig. 2 Biochemical conditions for in vitro PE-tag.

a, Schematic overview of quantification of 3′ flap generation by qRT-PCR at the HEK4 locus. HEK293T gDNA was treated with PE2 RNP to introduce the 3′ flap and then the editing efficiency was quantified by qRT-PCR with a tag-specific primer and a locus-specific primer. A pair of primers located ~2000 bp upstream of the target site serve as an internal control for gDNA normalization. b, HEK293T gDNA was treated with different concentrations of PE2 RNP to introduce the 3′ flap and then the editing efficiency was quantified by qRT-PCR. c, HEK293T gDNA was treated with 50 pmol of PE2 RNP to introduce the 3′ flap for different reaction times and then the editing efficiency was quantified by qRT-PCR. Results were obtained from three independent experiments and presented as mean ± SD. **** P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. d, HEK293T gDNA was treated with 50 pmol of PE2 RNP to introduce the 3′ flap in a buffer containing different concentrations of dNTPs and then the editing efficiency was quantified by qRT-PCR. Results were obtained from three independent experiments and presented as mean ± SD. **** P < 0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. e, HEK293T gDNA was treated with 50 pmol of PE2 RNP to introduce the 3′ flap at different reaction temperatures and then the editing efficiency was quantified by qRT-PCR. Results were obtained from three independent experiments and presented as mean ± SD. ** P < 0.01 and *** P < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. f, HEK293T gDNA was treated with 50 pmol of PE2 RNP to introduce the 3′ flap for two different reaction times (2 hrs and 24 hrs) and then the editing efficiency was quantified by qRT-PCR on target site and two OTs for the HEK4 site. Results were obtained from three independent experiments and presented as mean ± SD. **** P < 0.0001 by unpaired, two-tailed Student’s t-test.

Source data

Extended Data Fig. 3 The prime editing efficiency of 3′ flap generation with a series of pegRNAs.

a, The prime editing efficiency of 3′ flap generation with a series of pegRNAs which contain either one or two mismatches in the PBS region. HEK293T gDNA was treated with PE2 RNP containing the HEK4 20-7 pegRNA to introduce the 3′ flap for 2 hours, and then the flap incorporation efficiency was quantified by qRT-PCR with a tag-specific primer and a locus-specific primer. A pair of primers located ~2000 bp upstream of the target site serve as an internal control for data analysis. b, The efficiency of 3′ flap generation at HEK OT3 with a series of HEK4 20-7 pegRNAs which contain either one or two mismatches in the PBS region. HEK293T gDNA was treated with PE2 RNP to introduce the 3′ flap, and then the editing efficiency was quantified by qRT-PCR with a tag-specific primer and a locus-specific primer. A pair of primers located ~2000 bp upstream of the target site serve as an internal control for data analysis. Where shown, bar charts indicate the mean and error bars are s.d. of n = 3 independent qRT-PCR experiments.

Source data

Extended Data Fig. 4 in vitro PE-tag on purified gDNA.

a-b, Subset of potential off-target (OT) sites identified by in vitro PE-tag in PE2 RNP treated HEK293T gDNA at CDH4 locus (a) and VEGFA locus (b; Supplementary Data 1). Mismatches in the PBS and HA region of potential off-target sites relative to the target site (On) are shown in red and blue, respectively. UMI counts for each site are shown. c, Venn diagram of overlap between off-target sites discovered by in vitro PE-tag (UMI > 1) and previously described GUIDE-seq data for VEGFA site 211.

Source data

Extended Data Fig. 5 Prime editing at on target site and two off target sites in HEK293T cells.

a, (left) Comparison of precise editing efficiency for nucleotide substitution, targeted 1-bp deletion, and 1-bp insertion with PE2 at HEK4 (ON) target site and (right) indel rates at two off-target sites (OT-1 and OT-3) in HEK293T cells after co-transfecting pegRNA and PE2 expression plasmids. pegRNA sequence composition and type of sequence modification encoded is indicated in the legend, where the terminal numbers indicate the different HA lengths within the RTT. Frequencies of precise editing or indel rates were quantified by deep sequencing from PCR amplicons spanning each locus. Mock on target site editing represents all indels. Results were obtained from three independent experiments and presented as mean ± SD. *P < 0.05, ** P < 0.01 and *** P < 0.001 by unpaired, two-tailed Student’s t-test. To adjust for multiple comparisons, p-values were adjusted using the Benjamini-Hochberg (BH) method. b, indel rates for nucleotide substitution, targeted 1-bp deletion, and 1-bp insertion pegRNAs with PE2 at 6 additional potential off-target sites identified by PE-tag for HEK4 locus pegRNA in HEK293T cells. pegRNA sequence composition and type of sequence modification encoded is indicated in the legend, where the terminal numbers indicate the different HA lengths within the RTT. Frequencies of precise editing were quantified by deep sequencing from PCR amplicons spanning each locus. Results were obtained from three independent experiments and presented as mean ± SD. *P < 0.05, ** P < 0.01 and *** P < 0.001 by unpaired, two-tailed Student’s t-test. To adjust for multiple comparisons, p-values were adjusted using the Benjamini-Hochberg (BH) method.

Source data

Extended Data Fig. 6 Off target sites validation in HEK293T cells.

a, Indel rates for nucleotide substitution, targeted 1-bp deletion, and 1-bp insertion pegRNAs with PE2 at 8 potential off-target sites of top 20 OTs identified by GUIDE-seq that overlap with in vitro PE-tag for HEK4 locus pegRNA in HEK293T cells. Frequencies of editing were quantified by deep sequencing from PCR amplicons spanning each locus. Results were obtained from three independent experiments and presented as mean ± SD. *P < 0.05, ** P < 0.01 and *** P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test. b, Indel rates for nucleotide substitution, targeted 1-bp deletion, and 1-bp insertion pegRNAs with PE2 at 12 potential off-target sites of top 20 OTs identified by GUIDE-seq but absent in the in vitro PE-tag for HEK4 locus pegRNA in HEK293T cells. pegRNA sequence composition and type of sequence modification encoded is indicated in the legend, where the terminal numbers indicate the different HA lengths within the RTT. Frequencies of editing were quantified by deep sequencing from PCR amplicons spanning each locus. Results were obtained from three independent experiments and presented as mean ± SD. *P < 0.05, ** P < 0.01 and *** P < 0.001 by two-way ANOVA with Tukey’s multiple comparisons test. c, Editing outcomes with PE2 and 1-bp deletion pegRNA at HEK4 MISS-2 and MISS-7 in HEK293T cells. Frequencies of editing were quantified by deep sequencing of PCR amplicons spanning the locus. CRISPResso output shown for sequencing data.

Source data

Extended Data Fig. 7 Cas9 H840A and MMLV RT proteins are functionally independently in vitro in PE-tag system.

a, Schematic overview of the in vitro tag attachment in the human genome by purified PE2 or purified Cas9 H840A nickase and MMLV RT. gDNA is isolated from HEK293T cells and treated with indicated protein and a 20-7 pegRNA, resulting in a 20 bp tag attachment in the protospacer of on-target site. b, PE-tag was carried out in vitro on purified HEK293T gDNA with three different protein cocktails: 1) purified PE2 protein (Wolfe lab purified); 2) purified Cas9 H840A nickase and MMLV RT as separate proteins (Wolfe lab purified); 3) purified Cas9 H840A nickase (IDT) and MMLV RT (Thermofisher) as separate proteins using the HEK4 20-7 pegRNA for PE-tag. Locus specific primers (deep sequencing primer) were used to detect tag incorporation at the target site (on-target) and off-target site 3 (OT-3). All three systems were able to incorporate the sequencing tag into the target locus demonstrating that the MMLV RT can function in trans to the SpCas9 nickase for in vitro reactions. * indicates the expected PCR product size. Results were obtained from three independent experiments and representative results are shown. c, Venn diagram of overlap of PE potential off-target sites (UMI ≥ 1) discovered by three different protein cocktails. d, Subset of in vitro off-target (OT) sites identified. Mismatches in the PBS and HA region of potential off-target sites relative to the target site (On) are shown in red and blue, respectively. UMI counts for each site are shown.

Source data

Extended Data Fig. 8 PE-tag in HEK293T cells.

a and b, Subset of potential off-target (OT) sites identified by PE-tag using PE2 RNP or expression plasmid treated cells at VEGFA locus (a) and CDH4 locus (b; Supplementary Data 1). Mismatches in the PBS and HA region for potential off-target sites relative to the target site (On) are shown in red and blue, respectively. UMI counts for each site are shown for each treatment. c, Indel rates for tag insertion pegRNAs with PE2 at top 5 potential off-target sites identified by in vitro PE-tag for VEGFA locus pegRNA in HEK293T cells. Indel frequencies were quantified by deep sequencing from PCR amplicons spanning each locus. Results were obtained from three independent experiments and presented as mean ± SD. ** P < 0.01 and *** P < 0.001 by unpaired, two-tailed Student’s t-test. To adjust for multiple comparisons, p-values were adjusted using the Benjamini-Hochberg (BH) method.

Source data

Extended Data Fig. 9 Prime editing at a subset of off-target sites for three pathogenic correcting pegRNAs.

a, Venn diagram of overlap between potential off-target sites (UMI > 1) discovered by in vitro PE-tag and potential off-target sites discovered by GUIDE-tag in cell lines containing the pathogenic sequences treated with SpCas9 RNP and DSB tagging oligonucleotide. b-c, Comparison of editing rates by prime editor programmed with pegRNA to correct pathogenic sequence at a subset of potential OT sites identified by PE-tag in cells transfected with PE2 mRNA and pegRNA, plasmids expressing PE2 and pegRNA, or plasmids expressing PE2 and epegRNA at the CFTR locus (b) and MECP2 locus (c), respectively. Frequencies of editing rates were quantified by deep sequencing. Results were obtained from three independent experiments and presented as mean ± SD. *P < 0.05, ** P < 0.01 and *** P < 0.001 by unpaired, two-tailed Student’s t-test. To adjust for multiple comparisons, p-values were adjusted using the Benjamini-Hochberg (BH) method.

Source data

Extended Data Fig. 10 Dot plot of UMI count percentage.

Dot plot of UMI count percentage (UMI%) associated with the target site and discovered potential off-target sites for 5 pegRNAs (a) and 2 pegRNAs (b) analyzed by in vitro PE-tag, PE-tag in cells by PE2 plasmid delivery and PE-tag in cells by PE2 RNP or mRNA delivery. Red symbols indicate the target site. Blue or green symbols indicate the top off-target site with the remainder as black symbols. Source data file is provided.

Source data

Supplementary information

Supplementary Information

Supplementary Notes 1–6, Supplementary Figs. 1–12 and Supplementary Tables 1–4.

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Peer Review File

Supplementary Data 1

Off-target sites identified by PE-tag at different sites.

Source data

Source Data Figs. 1–5 and Extended Data Figs. 1–10

Statistical source data for Figs. 1–5 and Extended Data Fig. 1–10.

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Liang, SQ., Liu, P., Ponnienselvan, K. et al. Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag. Nat Methods 20, 898–907 (2023). https://doi.org/10.1038/s41592-023-01859-2

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