The DOI matches its Crossref record; 21 of the 21 references it cites were found in a registry or an index.
High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity
Open accessAbstract
The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting.
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“Moreover, in-depth studies on the outcomes of Cas9-induced double-strand break (DSB) repair have shown significant off-target effects, which resulted in substitution, insertion, deletion, and DNA translocation.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [12]
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Cites
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012) · Science · doi:10.1126/science.1225829 · Cited by 17,116
Self-citationMultiplex Genome Engineering Using CRISPR/Cas Systems
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., et al. (2013) · Science · doi:10.1126/science.1231143 · Cited by 15,339
Self-citationRNA-Guided Human Genome Engineering via Cas9
Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., & Church, G. M. (2013) · Science · doi:10.1126/science.1232033 · Cited by 9,210
Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
Qi, L. S., Larson, M. H., Gilbert, L. A., Doudna, J. A., Weissman, J. S., Arkin, A. P., & Lim, W. A. (2013) · Cell · doi:10.1016/j.cell.2013.02.022 · Cited by 5,016
Efficient genome editing in zebrafish using a CRISPR-Cas system
Hwang, W. Y., Fu, Y., Reyon, D., Maeder, M. L., Tsai, S. Q., Sander, J. D., Peterson, R. T., Yeh, J.-R. J., & Joung, J. K. (2013) · Nature Biotechnology · doi:10.1038/nbt.2501 · Cited by 2,937
RNA-guided editing of bacterial genomes using CRISPR-Cas systems
Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013) · Nature Biotechnology · doi:10.1038/nbt.2508 · Cited by 2,429
RNA-programmed genome editing in human cells
Jinek, M., East, A., Cheng, A., Lin, S., Ma, E., & Doudna, J. (2013) · eLife · doi:10.7554/elife.00471 · Cited by 2,098
Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
DiCarlo, J. E., Norville, J. E., Mali, P., Rios, X., Aach, J., & Church, G. M. (2013) · Nucleic Acids Research · doi:10.1093/nar/gkt135 · Cited by 1,566
Genetic engineering of human pluripotent cells using TALE nucleases
Hockemeyer, D. F., Wang, H., Kiani, S., Lai, C. S., Gao, Q., Cassady, J. P., Cost, G. J., Zhang, L., Santiago, Y., Miller, J. C., et al. (2011) · Nature Biotechnology · doi:10.1038/nbt.1927 · Cited by 1,161
Hockemeyer, D. F., Soldner, F., Beard, C., Gao, Q., Mitalipova, M. M., DeKelver, R. C., Katibah, G. E., Amora, R., Boydston, E. A., Zeitler, B., et al. (2009) · Nature Biotechnology · doi:10.1038/nbt.1562 · Cited by 1,064
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Detection of CRISPR/Cas9-Generated off-target effect by integration-defective lentiviral vector
Wang, X., Wu, Y., & Yee, J.-K. (2021) · Methods in molecular biology · doi:10.1007/978-1-0716-0687-2_14 · Cited by 5
Omura, S. N., Nakagawa, R., Kajimoto, S., Okazaki, S., Ishiguro, S., Mori, H., Onishi, K., Kashiwakura, Y., Hiramoto, T., Horinaka, K., et al. (2026) · Nature Communications · doi:10.1038/s41467-026-71626-2 · Cited by 2
Metadata
- Authors
- Pattanayak, V., Lin, S., Guilinger, J. P., Ma, E., Doudna, J. A., & Liu, D. R.
- Affiliations
- Howard Hughes Medical Institute (US)
- Corresponding author
- Doudna, Jennifer A
- Liu, David R
- Venue
- Nature Biotechnology
- Published
- 2013-08-11
- Volume
- 31
- Issue
- 9
- Pages
- 839-843
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3782611
- OpenAlex
- W2149402050
- Cited by
- 1,487
- References
- 21
- Citations by year
- 2026: 29 · 2025: 62 · 2024: 81 · 2023: 72 · 2022: 113 · 2021: 136 · 2020: 144 · 2019: 109 · 2018: 127 · 2017: 145 · 2016: 149 · 2015: 135 · 2014: 152 · 2013: 28
- FWCI
- 69.33
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of General Medical Sciences (R01GM073794, T32GM007753)
- 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 › RNA Interference and Gene Delivery
- Keywords
- CRISPR/Cas9, seed sequence, in vitro selection, genome editing, PAM sequence, HEK293T cells, human genome
- MeSH terms
- Bacterial Proteins, DNA, Endonucleases, Genetic Engineering, Humans, Streptococcus pyogenes, Genome, Sequence Analysis, DNA, Genomics, HEK293 Cells, RNA, Small Untranslated, High-Throughput Nucleotide Sequencing
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