Abstract

CRISPR–Cas genome-editing nucleases hold substantial promise for developing human therapeutic applications1,2,3,4,5,6 but identifying unwanted off-target mutations is important for clinical translation7. A well-validated method that can reliably identify off-targets in vivo has not been described to date, which means it is currently unclear whether and how frequently these mutations occur. Here we describe ‘verification of in vivo off-targets’ (VIVO), a highly sensitive strategy that can robustly identify the genome-wide off-target effects of CRISPR–Cas nucleases in vivo. We use VIVO and a guide RNA deliberately designed to be promiscuous to show that CRISPR–Cas nucleases can induce substantial off-target mutations in mouse livers in vivo. More importantly, we also use VIVO to show that appropriately designed guide RNAs can direct efficient in vivo editing in mouse livers with no detectable off-target mutations. VIVO provides a general strategy for defining and quantifying the off-target effects of gene-editing nucleases in whole organisms, thereby providing a blueprint to foster the development of therapeutic strategies that use in vivo gene editing.

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Data availability

Sequence data that support the findings of this study have been deposited with SRA accession number SRP151131. All other data that support the findings of this study are available from the corresponding authors upon reasonable request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

J.K.J. is supported by the Desmond and Ann Heathwood MGH Research Scholar Award. J.K.J., M.L.B. and J.A.G. were supported by a sponsored research agreement with AstraZeneca. L.P. is supported by a National Human Genome Research Institute (NHGRI) Career Development Award (R00HG008399). J.K.J., M.J.A. and J.M.-L. are supported by a National Institutes of Health Maximizing Investigators' Research Award (MIRA) (R35 GM118158). J.K.J., L.P. and K.C. are supported by the Defense Advanced Research Projects Agency (HR0011-17-2-0042). We thank M. Snowden, S. Platz and S. Rees for resource allocation from AstraZeneca Research Funds. We thank J. Y. Hsu for discussions and input.

Reviewer information

Nature thanks F. Urnov and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

Author notes

    • Alba Carreras

    Present address: Wallenberg Laboratory and Sahlgrenska Center for Cardiovascular and Metabolic Research, Department of Molecular and Clinical Medicine, University of Gothenburg, Gothenburg, Sweden

    • Tania Baccega

    Present address: San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy

    • Shengdar Q. Tsai

    Present address: Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    • Lorenz M. Mayr

    Present address: GE Healthcare Life Sciences, The Grove Centre, Amersham, UK

  1. These authors contributed equally: Pinar Akcakaya, Maggie L. Bobbin

Affiliations

  1. Discovery Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden

    • Pinar Akcakaya
    • , Michelle J. Porritt
    • , Alba Carreras
    • , Tania Baccega
    • , Mikael Bjursell
    • , Lorenz M. Mayr
    • , Mohammad Bohlooly-Y
    •  & Marcello Maresca
  2. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA, USA

    • Maggie L. Bobbin
    • , Jimmy A. Guo
    • , Jose Malagon-Lopez
    • , Kendell Clement
    • , Sara P. Garcia
    • , Shengdar Q. Tsai
    • , Nhu T. Nguyen
    • , Luca Pinello
    • , Martin J. Aryee
    •  & J. Keith Joung
  3. Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA, USA

    • Maggie L. Bobbin
    • , Jimmy A. Guo
    • , Jose Malagon-Lopez
    • , Kendell Clement
    • , Shengdar Q. Tsai
    • , Nhu T. Nguyen
    •  & J. Keith Joung
  4. Department of Pathology, Harvard Medical School, Boston, MA, USA

    • Maggie L. Bobbin
    • , Jose Malagon-Lopez
    • , Kendell Clement
    • , Shengdar Q. Tsai
    • , Luca Pinello
    • , Martin J. Aryee
    •  & J. Keith Joung
  5. Advanced Medicines Safety, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Cambridge, UK

    • Mick D. Fellows
  6. Quantitative Biology, Discovery Sciences, IMED Biotech Unit, AstraZeneca, Cambridge, UK

    • Mike A. Firth
  7. Pathology Science, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden

    • Frank Seeliger
  8. Advanced Medicines Safety, Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden

    • Roberto Nitsch

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Contributions

P.A., M.J.P., T.B. and M.B. executed intravenous tail vein injections. P.A. and T.B. coordinated vena saphena blood sampling and performed plasma extractions. P.A., M.J.P., A.C., T.B., M.B., M.M. and R.N. performed in vivo terminations and organ collection. P.A. performed Surveyor assays for genomic DNAs of mouse livers. P.A. and T.B. performed ELISA for Pcsk9 protein detection in plasma samples. M.L.B, J.A.G., S.Q.T. and N.T.N. performed the CIRCLE-seq and targeted amplicon sequencing experiments. J.M.-L., K.C., S.P.G., L.P. and M.J.A. performed bioinformatic and computational analysis of the off-target experiments. M.A.F. generated AstraZeneca proprietary software for gRNA identification. P.A. designed gRNAs with help of M.A.F., and validated their functional activity. F.S. performed mouse phenotypic characterization. P.A., M.L.B., S.Q.T., M.M., M.B.-Y, A.C., R.N., M.D.F., L.M.M. and J.K.J. conceived of and designed the study. P.A., M.L.B., M.M. and J.K.J. organized and supervised experiments. P.A., M.L.B., J.A.G., M.M. and J.K.J. prepared the manuscript with input from all authors.

Competing interests

J.K.J. has financial interests in Beam Therapeutics, Blink Therapeutics, Editas Medicine, Endcadia, Monitor Biotechnologies (formerly known as Beacon Genomics), Pairwise Plants, Poseida Therapeutics and Transposagen Biopharmaceuticals. M.J.A. and S.Q.T. have financial interests in Monitor Biotechnologies. J.K.J.’s and M.J.A.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. S.Q.T. and J.K.J. are co-inventors on a patent describing the CIRCLE-seq method. P.A., M.D.F., M.J.P., M.A.F., F.S., M.B., R.N., M.B.Y. and M.M. are employees and shareholders of AstraZeneca. L.M.M. is an employee and shareholder of GE Healthcare and a shareholder of AstraZeneca.

Corresponding authors

Correspondence to Marcello Maresca or J. Keith Joung.

Extended data figures and tables

  1. Extended Data Fig. 1 gP–Cas9 efficiently mutates the mouse Pcsk9 gene and reduces levels of Pcsk9 protein in plasma in vivo.

    a, gP was designed to target a sequence within exon 1 of the mouse Pcsk9 gene that has many closely related genomic sites (that is, those with 1–3 mismatches relative to the on-target site; Extended Data Table 1). Blue bars indicate exons for the mouse genomic region. b, Surveyor assay and next-generation DNA sequencing data demonstrate efficient in vivo modification of the on-target mouse Pcsk9 gene site in mouse liver by gP–Cas9. Assays were performed on day 4 and on week 3 after the administration of adenoviral vectors that encode gP–Cas9 (gP) or negative control GFP–Cas9 (GFP). For each time point, the assays used genomic DNA isolated from livers of n = 3 biologically independent wild-type C57BL/6N (WT) mice or C57BL/6N-derived mice containing a single copy of the human PCSK9 open reading frame under albumin promoter, knocked into the Rosa26 locus (KI). Asterisks indicate the cleaved PCR products expected after treatment with Surveyor nuclease. Percentages show the frequencies of indel mutations determined by targeted amplicon sequencing using next-generation sequencing; these are the same values shown for the on-target site in Fig. 1b. Lines divide lanes taken from different locations on the same gel. For source data for Surveyor assays and targeted amplicon sequencing, see Supplementary Fig. 1 and Supplementary Table 2, respectively. c, Mouse Pcsk9 protein levels in plasma measured in n = 3 biologically independent wild-type and knock-in mice and human PCSK9 protein levels in plasma measured in n = 3 biologically independent knock-in mice, after nuclease treatment. Protein levels were assessed on day 4, 7 and week 3 after administration of gP or control GFP adenoviral vectors and normalized to baseline levels. Significant differences between experimental and control groups were determined using two-way ANOVA and Sidak’s two-sided adjusted multiple comparisons test; **P < 0.01, ***P < 0.001, ****P < 0.0001. See Source Data for Extended Data Fig. 4b for exact adjusted P values. All values are presented as group means, and error bars represent standard error of the mean. The enhanced reduction of levels of Pcsk9 in plasma relative to the frequency of observed Pcsk9 genetic alteration is consistent with previously published studies (Supplementary Discussion). Source data

  2. Extended Data Fig. 2 Bio-distribution studies of adenovirus-serotype 5 in mice.

    a, Schematic of integrated reporter construct in R26R mice used to assess delivery of Cre recombinase using adenovirus serotype 5 vector. Cre-mediated excision of a loxP-flanked transcriptional stop signal upstream of a lacZ gene results in expression of β-galactosidase enzyme. β-Galactosidase expression can be quantified by staining dissected tissues with X-gal, a compound that turns blue when cleaved by this enzyme. b, Quantification of β-galactosidase expression in sections of various dissected organs from n = 2 biologically independent R26R mice intravenously injected with adenovirus serotype 5 vector encoding Cre. Matched organs sections from a R26R mouse intravenously injected with an adenovirus serotype 5 vector encoding GFP were used to determine background staining levels and serve as a negative control. Matched organ sections from Z/EG mice that constitutively express lacZ (β-galactosidase) and intravenously injected with PBS (rather than adenovirus) were used to provide positive staining controls. All mice were evaluated one week after adenovirus or PBS injection. The experiment was performed once.

  3. Extended Data Fig. 3 Breeding strategy for generating experimental mice containing human PCSK9 open reading frame knocked into the Rosa26 locus.

    C57BL/6N-derived mouse line containing a single copy of the human PCSK9 open reading frame knocked into the Rosa26 locus (C57BL/6N hPCSK9KI+/−) are used for breeding with C57BL/6N mice. Offspring yielded experimental animals that are C57BL/6N hPCSK9KI+/− (referred to as knock-in) and C57BL/6N hPCSK9KI−/− (referred to as wild type) males.

  4. Extended Data Fig. 4 Scatter plot of CIRCLE-seq read counts for sites identified with gP–Cas9 on genomic DNA from n = 1 wild-type and n = 1 knock-in mice.

    Read counts are shown on a logarithmic scale and colours indicate the number of mismatches in each off-target site relative to the on-target site. Sites shown as triangles were chosen for targeted amplicon sequencing. The correlation r2 value obtained using all values in the scatter plot is shown in the upper left-hand corner and was obtained using a linear regression.

  5. Extended Data Fig. 5 Comparison of closely matched sites identified in silico and off-target cleavage sites identified by CIRCLE-seq.

    Venn diagrams comparing off-target cleavage sites in mouse genomic DNA identified by CIRCLE-seq experiments with closely matched sites (up to six mismatches relative to the on-target site) in the mouse genome identified in silico by Cas-OFFinder are shown for the Cas9 gRNAs gP, gM and gMH.

  6. Extended Data Fig. 6 Genetic and phenotypic alterations induced by delivery of gM–Cas9 and gMH–Cas9 in vivo.

    a, Sequence and location of the Cas9 gM (mouse) and gMH (mouse and human) target sites in the endogenous mouse Pcsk9 gene and human PCSK9 transgene inserted at the mouse Rosa26 locus. The single base position that differs between the gMH target sites in the mouse Pcsk9 gene and the human PCSK9 transgene is highlighted in red. Blue bars indicate exons for the mouse genomic region and purple bars represent exons for the human genomic locus; the PAM sequence for the sites is in bold and the spacer sequence is underlined. b, Surveyor assay and next-generation DNA sequencing data demonstrate efficient in vivo modification of the on-target endogenous mouse Pcsk9 site and human PCSK9 transgene in mouse liver. Assays were performed at day 4 and at week 3 after administration of adenoviral vectors that encode gM and Cas9 (gM), gMH and Cas9 (gMH) or GFP and Cas9 (GFP) using genomic DNA isolated from livers of n = 3 biologically independent wild-type and knock-in mice. Asterisks indicate the cleaved PCR products expected following treatment with Surveyor nuclease. Percentages show the frequencies of indel mutations determined by targeted amplicon sequencing using next-generation sequencing; these are the same values shown for the on-target sites in Fig. 3 and Extended Data Fig. 7. Lines divide lanes taken from different locations on the same gel. For source data for Surveyor assays, see Supplementary Fig. 1. For source data for targeted amplicon sequencing, see Supplementary Tables 6 and 7 for gM and gMH, respectively. b, Mouse Pcsk9 protein levels measured in plasma in n = 3 biologically independent wild-type and knock-in mice, and human PCSK9 protein levels measured in plasma in n = 3 biologically independent knock-in mice after CRISPR–Cas nuclease treatment. Protein levels in plasma were assessed at day 4, 7 and week 3 after the administration of gM, gMH or control GFP adenoviral vectors and normalized to baseline levels at each time point. Significant differences between groups were determined using two-way ANOVA and Dunnett’s two-sided adjusted multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Source Data for exact adjusted P values. Values are presented as group means, error bars represent standard errors of the mean. Source data

  7. Extended Data Fig. 7 Assessment of in vivo off-target indel mutations induced by gMH–Cas9.

    Indel mutation frequencies determined by targeted amplicon sequencing (using high-throughput sequencing) are presented as heat maps for the gMH–Cas9 on-target site (black square) and 63 off-target sites identified from CIRCLE-seq experiments. Each locus was assayed in n = 3 biologically independent mice (labelled 1, 2 and 3) using genomic DNA isolated from the liver of wild-type and knock-in mice treated with experimental adenoviral vector that encodes gMH–Cas9 (gRNA +) or control adenoviral vector GFP–Cas9 (gRNA −). For each site, mismatches relative to the on-target site are shown with coloured boxes and bases in the spacer sequence and are numbered from 1 (most proximal to the PAM) to 20 (most distal from the PAM). The number of read counts found for each site from the CIRCLE-seq experiments on wild-type and knock-in mouse genomic DNA are shown in the left columns (ranked from highest to lowest based on counts in the wild-type genomic DNA CIRCLE-seq experiment). Each box in the heat map represents a single sequencing experiment. Sites that were significantly different between the experimental (gRNA +) and control (gRNA −) samples are highlighted with an orange outline around the boxes. Additional closely matched sites in the mouse genome (not identified from the CIRCLE-seq experiments) that were examined for indel mutations are boxed in red at the bottom of the figure. See Supplementary Table 7 for source data and P values (negative binomial).

  8. Extended Data Table 1 Numbers of off-target sites for gP, gM and gMH identified by Cas-OFFinder (in silico) and CIRCLE-seq (experimental)
  9. Extended Data Table 2 Off-target sites identified by CIRCLE-seq for gP, gM and gMH that exhibit single nucleotide polymorphisms or indels based on CIRCLE-seq data
  10. Extended Data Table 3 Numbers of closely matched sites in the mouse genome with canonical NGG, alternate NAG and other alternate non-NGG or non-NAG PAMs for gP, gM and gMH

Supplementary information

  1. Supplementary Information

    This file contains a Supplementary Discussion. Additional discussion presented includes review of previous studies examining off-target mutations in vivo, potential explanations for relatively greater reductions in plasma Pcsk9 compared to efficiencies of Pcsk9 gene mutations observed, description of how VIVO might be used with CRISPR-Cas variants and orthologues, other gene-editing platforms/delivery methods, and non-mammalian organisms.

  2. Reporting Summary

  3. Supplementary Figures

    This file contains uncropped gels for extended data figures.

  4. Supplementary Table

    This file contains Supplementary Table 1: Off-target cleavage sites identified by CIRCLE-seq for gP/SpCas9 nuclease. Chromosomal coordinates are provided, followed by the CIRCLE-seq read count for off-target sites found in WT mouse genomic DNA, the off-target sequence, number of mismatches, bulge mismatch sequence, the score of the number of mismatches with bulges, and the CIRCLE-seq read count for off-targets identified in KI mouse genomic DNA.

  5. Supplementary Table

    This file contains Supplementary Table 2: Potential off-target sites for gP/SpCas9 analyzed by targeted amplicon deep sequencing from the livers of WT and KI mice treated with adenoviral vectors. Chromosomal coordinates are provided, followed by MiSeq run ID and the FASTQ files used, sample information including tissue type, time point, treatment and replicate. The next columns detail the read counts for each sample and statistical test results between the treatment groups, followed by primers used. NB: Negative binomial test.

  6. Supplementary Table

    This file contains Supplementary Table 3: Off-target cleavage sites identified by CIRCLE-seq for SpCas9 and the gM gRNA. Chromosomal coordinates are provided followed by the CIRCLE-seq read count for off-target sites found in WT mouse genomic DNA, the off-target sequence, number of mismatches, bulge mismatch sequence, the score of the number of mismatches with bulges, and the CIRCLE-seq read count for off-targets identified in KI mouse genomic DNA.

  7. Supplementary Table

    This file contains Supplementary Table 4: Off-target cleavage sites identified by CIRCLE-seq for SpCas9 and the gMH gRNA. Chromosomal coordinates are provided followed by the CIRCLE-seq read count for off-target sites found in WT mouse genomic DNA, the off-target sequence, number of mismatches, bulge mismatch sequence, the score of the number of mismatches with bulges, and the CIRCLE-seq read count for off-targets identified in KI mouse genomic DNA.

  8. Supplementary Table

    This file contains Supplementary Table 5: Potential off-target sites for gM/SpCas9 identified by CIRCLE-seq with no reads in either WT or KI untreated mice analyzed by targeted amplicon deep sequencing to assess potential single nucleotide polymorphisms. Numbers and percentages of total sequence reads that differ from the expected off-target sites are shown. The "differences in WT vs KI percentage" column shows absolute values of the difference between these reads. Highest difference found was 7.5%, suggesting SNPs do not explain differential read counts observed compared with CIRCLE-seq.

  9. Supplementary Table

    This file contains Supplementary Table 6: Potential off-target sites for gM/SpCas9 analyzed by targeted amplicon deep sequencing from the livers of WT and KI mice treated with adenoviral vectors. Chromosomal coordinates are provided, followed by MiSeq run ID and the FASTQ files used, sample information including tissue type, time point, treatment and replicate. The next columns detail the read counts for each sample and statistical test results between the treatment groups, followed by primers used. NB: Negative binomial test.

  10. Supplementary Table

    This file contains Supplementary Table 7: Potential off-target sites for gMH/SpCas9 analyzed by targeted amplicon deep sequencing from the livers of WT and KI mice treated with adenoviral vectors. Chromosomal coordinates are provided, followed by MiSeq run ID and the FASTQ files used, sample information including tissue type, time point, treatment and replicate. The next columns detail the read counts for each sample and statistical test results between the treatment groups, followed by primers used. NB: Negative binomial.

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https://doi.org/10.1038/s41586-018-0500-9

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