Digenome-seq: Genome-wide profiling of CRISPR-cas9 off-target effects in human cells
Abstract
Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.
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Digenome-seq is a highly sensitive method that can identify indels with 0.1% frequency or lower.
“Digenome-seq is a highly sensitive method that can identify indels with 0.1% frequency or lower (Kim et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Kim et al., 2015
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Towards a new era in medicine: therapeutic genome editing
Porteus, M. H. (2015) · Genome Biology · doi:10.1186/s13059-015-0859-y · Cited by 69 · see citing works
Genome-wide detection of CRISPR editing in vivo using GUIDE-tag
Liang, S.-Q., Liu, P., Smith, J. L., Mintzer, E., Maitland, S., Dong, X., Yang, Q., Lee, J., Haynes, C. M., Zhu, L. J., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-28135-9 · Cited by 54 · see citing works
Jo, D. H., Koo, T., Cho, C. S., Kim, J. H., Kim, J.-S., & Kim, J. H. (2019) · Molecular Therapy · doi:10.1016/j.ymthe.2018.10.009 · Cited by 57 · see citing works
Self-citationConcerns regarding ‘off-target’activity of genome editing endonucleases
Kadam, U. S., Shelake, R. M., Chavhan, R. L., & Suprasanna, P. (2018) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2018.03.027 · Cited by 47 · see citing works
Massively targeted evaluation of therapeutic CRISPR off-targets in cells
Pan, X., Qu, K., Yuan, H., Xiang, X., Anthon, C., Pashkova, L., Liang, X., Han, P., Corsi, G. I., Xu, F., et al. (2022) · Nature Communications · doi:10.1038/s41467-022-31543-6 · Cited by 42 · see citing works
crisprSQL: a novel database platform for CRISPR/Cas off-target cleavage assays
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Self-citationLearning to quantify uncertainty in off-target activity for CRISPR guide RNAs
Özden, F., & Minary, P. (2024) · Nucleic Acids Research · doi:10.1093/nar/gkae759 · Cited by 21 · see citing works
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Genome-wide CRISPR off-target DNA break detection by the BLISS method
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CRISPR off-target analysis platforms
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Evolution of prime editing systems: move forward to the treatment of hereditary diseases
Volodina, O. V., Fabrichnikova, A. R., Anuchina, A. A., Mishina, O. S., Lavrov, A. V., & Smirnikhina, S. A. (2025) · Current Gene Therapy · doi:10.2174/0115665232295117240405070809 · Cited by 1 · see citing works
- Authors
- Kim, D., Bae, S., Park, J., Kim, E., Kim, S., Yu, H. R., Hwang, J., Kim, J.-I., & Kim, J.-S.
- Affiliations
- Seoul National University (KR)
- ToolGen (South Korea) (KR)
- Corresponding author
- Kim, Jong-Il
- ORCID
- Venue
- Nature Methods
- Published
- 2015-02-09
- Volume
- 12
- Issue
- 3
- Pages
- 237-243
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- OpenAlex
- W2085993152
- Cited by
- 992
- References
- 48
- Citations by year
- 2026: 51 · 2025: 60 · 2024: 67 · 2023: 58 · 2022: 85 · 2021: 100 · 2020: 116 · 2019: 99 · 2018: 106 · 2017: 100 · 2016: 104 · 2015: 46
- FWCI
- 51.55
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- The Royal Society of Chemistry’s Journals, Books and Databases (The Royal Society of Chemistry) (submittedVersion)
- Seoul National University Open Repository (Seoul National University) (submittedVersion)
- Funders
- Institute for Basic Science
- 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
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CRISPR/Cas9, off-target effects, single guide RNA, genome-wide characterization, indels, human cells, programmable nucleases
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Cell Line, Endonucleases, Haploidy, Humans, Mutation, Sensitivity and Specificity, Reproducibility of Results, Genome, Human, RNA, Guide, Kinetoplastida, Limit of Detection, High-Throughput Nucleotide Sequencing, Clustered Regularly Interspaced Short Palindromic Repeats
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