Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq
Open accessPublished version of a preprint
Abstract
CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.
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RNP electroporation for Cas9/sgRNA delivery in cell culture eliminates off-target effects.
“| Delivery methods improvement | Application in cell culture [89–94] | RNP electroporation: Rapid transfection and turnover | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [90]
The Introduction (page unavailable) reports editing patient-derived iPSCs with HiFi-Cas9 RNPs and detecting two off-targets with the wild-type guide. That contradicts the claim that RNP delivery eliminates off-target effects; the passage does not specify electroporation or rapid turnover. (LLM, conf=0.86)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measuredViral transduction for Cas9/sgRNA delivery in cell culture may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in cell culture [89–94] | Viral transduction | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [90]
The Introduction reports off-target indels after adenoviral delivery of Cas9 and a guide RNA in mice, and separately reports off-target editing in cultured cells. It does not show that viral delivery in cell culture causes off-target mutations to accumulate over time. (LLM, conf=0.82)
Experimental — accuracy not yet measuredDiscover-seq is broadly applicable for various types of tissue or cell samples.
“Because good ChIP-grade MRE11 antibodies are available and there is no need to deliver any exogenous components to the body, Discover-seq is broadly applicable for various types of tissue or cell samples (Wienert et al., 2019; Cromer et al., 2023).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Wienert et al., 2019
Discover-seq utilizes MRE11, an endogenous DNA repair protein, to identify CRISPR-Cas-induced DSBs in vivo.
“Discover-seq utilizes MRE11, an endogenous DNA repair protein, to identify CRISPR-Cas-induced DSBs in vivo (Wienert et al., 2019).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Wienert et al., 2019
Discover-seq only captures DSBs that are present at the moment of sample preparation, so its sensitivity should be carefully assessed, concerning a potentially high false negative rate.
“However, because MRE11-DSB binding is transient, Discover-seq only captures DSBs that are present at the moment of sample preparation (Wienert et al., 2019). Therefore, the sensitivity of Discover-seq should be carefully assessed, concerning a potentially high false negative rate.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Wienert et al., 2019
Plasmid transfection for Cas9/sgRNA delivery in cell culture may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in cell culture [89–94] | Plasmid transfection | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [90]
The Introduction (page unavailable) reports Cas9 off-targets in edited cells, including iPSCs edited with Cas9 ribonucleoproteins. Searches for plasmid transfection and accumulation of off-target mutations found no passage linking that delivery method to the claimed effect. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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- Authors
- Wienert, B., Wyman, S. K., Richardson, C. D., Yeh, C. D., Akcakaya, P., Porritt, M. J., Morlock, M., Vu, J. T., Kazane, K. R., Watry, H. L., Judge, L. M., Conklin, B. R., Maresca, M., & Corn, J. E.
- Affiliations
- Gladstone Institutes (US)
- Innovative Genomics Institute (US)
- Innovative Genomics Institute, University of California Berkeley, Berkeley, CA 94704, USA.
- ETH Zurich (CH)
- Discovery Biology, AstraZeneca, 43150 Gothenburg, Sweden.
- AstraZeneca (Sweden) (SE)
- Corresponding author
- Corn, Jacob E.
- ORCID
- Venue
- Science
- Published
- 2019-04-19
- Volume
- 364
- Issue
- 6437
- Pages
- 286-289
- ISSN
- 0036-8075 (ISSN-L)
- 1095-9203
- PMC
- PMC6589096
- OpenAlex
- W2900885456
- Cited by
- 434
- References
- 39
- Citations by year
- 2026: 53 · 2025: 49 · 2024: 40 · 2023: 46 · 2022: 66 · 2021: 69 · 2020: 81 · 2019: 29
- FWCI
- 20.26
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Science (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- National Heart, Lung, and Blood Institute (DP2-HL-141006, R01-HL130533, R01-HL13535801)
- National Eye Institute (R01-EY028249)
- California Institute for Regenerative Medicine (TRAN1-09292)
- Li Ka Shing Foundation
- Heritage Medical Research Institute
- American Australian Association (Sir Keith Murdoch Fellowship)
- NHMRC (Early Career Fellowship)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Keywords
- DNA repair proteins, single-base resolution, genome editing, guide RNA, induced pluripotent stem cells, adenoviral gene therapy, therapeutic genome editing
- MeSH terms
- Gene Editing, MRE11 Homologue Protein, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Adenoviridae, Animals, Cell Line, DNA, DNA Repair, Humans, RNA, Guide, Kinetoplastida, Sequence Analysis, DNA, K562 Cells, DNA Repair Enzymes, Chromatin Immunoprecipitation, DNA Breaks, Double-Stranded, Induced Pluripotent Stem Cells, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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