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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Citations
Cites
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Self-citationMultiplex Genome Engineering Using CRISPR/Cas Systems
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Self-citationRNA-Guided Human Genome Engineering via Cas9
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Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression
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Efficient genome editing in zebrafish using a CRISPR-Cas system
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Genome engineering using the CRISPR-Cas9 system
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., & Zhang, F. (2013) · Nature Protocols · doi:10.1038/nprot.2013.143 · Cited by 11,133
The new frontier of genome engineering with CRISPR-Cas9
Doudna, J. A., & Charpentier, E. (2014) · Science · doi:10.1126/science.1258096 · Cited by 7,062
“The amount of Cas9 enzyme expressed in the cell is an important factor in tolerance to mismatches (138).”
Self-citationDevelopment and Applications of CRISPR-Cas9 for Genome Engineering
Hsu, P. D., Lander, E. S., & Zhang, F. (2014) · Cell · doi:10.1016/j.cell.2014.05.010 · Cited by 5,762
“, 2013), these studies collectively demonstrate that Cas9 tolerates mismatches throughout the guide sequence in a manner that is sensitive to the number, position, and distribution of the mismatches (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013b; Pattanayak et al., 2013).”
InfluentialProgrammable editing of a target base in genomic DNA without double-stranded DNA cleavage
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A., & Liu, D. R. (2016) · Nature · doi:10.1038/nature17946 · Cited by 5,437
Self-citationDouble nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y., et al. (2013) · Cell · doi:10.1016/j.cell.2013.08.021 · Cited by 3,299
“We and others have previously shown that, although singlebase mismatches between the PAM-distal region of the guide sequence and target DNA are well tolerated by Cas9, multiple mismatches in this region can significantly affect on-target activity (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al…”
Background
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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