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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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.
“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]
The paper’s results (indexed under Abstract; page unavailable) report Cas9-induced cleavage and evidence of non-homologous end joining at five of 49 tested off-target sites in HEK293T cells. They do not establish the claimed full range of repair outcomes—substitution, insertion, deletion, and DNA translocation. The mutations described relative to intended target sites are pre-existing sequence differences, not reported repair outcomes. (LLM, conf=0.87)
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Long, C., McAnally, J. R., Shelton, J. M., Mireault, A. A., Bassel-Duby, R., & Olson, E. N. (2014) · Science · doi:10.1126/science.1254445 · Cited by 720 · see citing works
Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection
Liang, X., Potter, J., Kumar, S., Zou, Y., Quintanilla, R., Sridharan, M., Carte, J., Chen, W., Roark, N., Ranganathan, S., et al. (2015) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2015.04.024 · Cited by 733 · see citing works
Genome-editing Technologies for Gene and Cell Therapy
Maeder, M. L., & Gersbach, C. A. (2016) · Molecular Therapy · doi:10.1038/mt.2016.10 · Cited by 722 · see citing works
Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases
Frock, R. L., Hu, J., Meyers, R. M., Ho, Y.-J., Kii, E., & Alt, F. W. (2015) · Nature Biotechnology · doi:10.1038/nbt.3101 · Cited by 673 · see citing works
CRISPR/Cas9 for genome editing: progress, implications and challenges
Zhang, F., Wen, Y., & Guo, X. (2014) · Human Molecular Genetics · doi:10.1093/hmg/ddu125 · Cited by 694 · see citing works
“The dosage of CRISPR/Cas9 is another factor affecting off-target mutations and should be carefully controlled (29,78).”
BackgroundLin, Y., Cradick, T. J., Brown, M. T., Deshmukh, H., Ranjan, P., Sarode, N., Wile, B. M., Vertino, P. M., Stewart, F. J., & Bao, G. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku402 · Cited by 634 · see citing works
“For example, the cleavage activity induced by the R-01 variant at position 2/1 may be alternatively interpreted as Cas9 cleavage with a GTG PAM (Figure 2C and Supplementary Figure S1), which is highly unlikely according to previous studies (20,21).”
Targeted Mutagenesis in Zea mays Using TALENs and the CRISPR/Cas System
Liang, Z., Zhang, K., Chen, K., & Gao, C. (2013) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/j.jgg.2013.12.001 · Cited by 662 · see citing works
Feng, Z., Mao, Y., Xu, N., Zhang, B., Wei, P., Yang, D., Wang, Z., Zhang, Z., Zheng, R., Yang, L., et al. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1400822111 · Cited by 657 · see citing works
“Previous studies in human cell lines indicated a high frequency of off-target effect of CRISPR/Cas-induced mutagenesis (12, 13) but a lower off-target effect in mice and zebrafish (14, 15).”
BackgroundGene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA
Ramakrishna, S., Kwaku Dad, A.-B., Beloor, J., Gopalappa, R., Lee, S.-K., & Kim, H. (2014) · Genome Research · doi:10.1101/gr.171264.113 · Cited by 655 · see citing works
“…effect) and specificity (off-target effect) are strongly affected by Cas9 protein and guide RNA concentrations (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013), and (2) it is challenging to exactly control these concentrations so that they are the same in the CPP-mediated direct…”
BackgroundSelf‐assembled DNA nanoclews for the efficient delivery of CRISPR–Cas9 for genome editing
Sun, W., Ji, W., Hall, J. M., Hu, Q., Wang, C., Beisel, C. L., & Gu, Z. (2015) · Angewandte Chemie International Edition · doi:10.1002/anie.201506030 · Cited by 629 · see citing works
Correction of a genetic disease in mouse via use of CRISPR-Cas9
Wu, Y., Liang, D., Wang, Y., Bai, M., Tang, W., Bao, S., Yan, Z., Li, D., & Li, J. (2013) · Cell stem cell · doi:10.1016/j.stem.2013.10.016 · Cited by 614 · see citing works
Poirot, L., Philip, B., Schiffer-Mannioui, C., Le Clerre, D., Chion-Sotinel, I., Derniame, S., Potrel, P., Bas, C., Lemaire, L., Galetto, R., et al. (2015) · Cancer Research · doi:10.1158/0008-5472.can-14-3321 · Cited by 605 · see citing works
Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a
Swarts, D. C., John van der, O. O. S. T., & Jínek, M. (2017) · Molecular Cell · doi:10.1016/j.molcel.2017.03.016 · Cited by 615 · see citing works
“, 2011), although mismatches to seed sequence nucleotides are sometimes tolerated (Fu et al., 2013, 2014; Kleinstiver et al., 2016; Pattanayak et al., 2013).”
BackgroundPast, present, and future of CRISPR genome editing technologies
Pacesa, M., Pelea, O., & Jinek, M. (2024) · Cell · doi:10.1016/j.cell.2024.01.042 · Cited by 609 · see citing works
Belhaj, K., Chaparro‐Garcia, A., Kamoun, S., & Nekrasov, V. (2013) · Plant Methods · doi:10.1186/1746-4811-9-39 · Cited by 582 · see citing works
“Recently, a number of reports have systematically examined specificity of the CRISPR/Cas system in human cells as well as in vitro [26-30].”
BackgroundHighly Improved Gene Targeting by Germline-Specific Cas9 Expression in Drosophila
Kondo, S., & Ueda, R. (2013) · Genetics · doi:10.1534/genetics.113.156737 · Cited by 563 · see citing works
“Recent studies have shown that Cas9 accommodates multiple mismatches in the 20-bp target sequence, raising concerns about off-target effect (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”
BackgroundMultiplexed CRISPR technologies for gene editing and transcriptional regulation
McCarty, N. S., Graham, A. E., Studená, L., & Ledesma‐Amaro, R. (2020) · Nature Communications · doi:10.1038/s41467-020-15053-x · Cited by 547 · see citing works
Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents
Gao, X., Tao, Y., Lamas, V., Huang, M., Yeh, W.-H., Pan, B., Hu, Y.-J., Hu, J. H., Thompson, D. B., Shu, Y., et al. (2018) · Nature · doi:10.1038/nature25164 · Cited by 536 · see citing works
Genome Engineering with Targetable Nucleases
Carroll, D. (2014) · Annual Review of Biochemistry · doi:10.1146/annurev-biochem-060713-035418 · Cited by 541 · see citing works
Editing plant genomes with CRISPR/Cas9
Belhaj, K., Chaparro‐Garcia, A., Kamoun, S., Patron, N. J., & Nekrasov, V. (2014) · Current Opinion in Biotechnology · doi:10.1016/j.copbio.2014.11.007 · Cited by 541 · see citing works
Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9
Gupta, R. M., & Musunuru, K. (2014) · Journal of Clinical Investigation · doi:10.1172/jci72992 · Cited by 545 · see citing works
Andersson, M., Turesson, H., Nicolia, A., Fält, A., Samuelsson, M., & Hofvander, P. (2016) · Plant Cell Reports · doi:10.1007/s00299-016-2062-3 · Cited by 540 · see citing works
“It has also been shown that the strength of the promoter can have an effect on target specificity, and a high concentration of sgRNACas9 transcripts can increase off-target mutations (Hsu et al. 2013; Pattanayak et al. 2013).”
BackgroundRNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection
Hu, W., Kaminski, R., Yang, F., Zhang, Y., Cosentino, L., Li, F., Luo, B., Alvarez-Carbonell, D., Garcia-Mesa, Y., Karn, J., et al. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1405186111 · Cited by 527 · see citing works
Genome Engineering Using CRISPR-Cas9 System
Cong, L., & Zhang, F. (2014) · Methods in molecular biology · doi:10.1007/978-1-4939-1862-1_10 · Cited by 518 · see citing works
Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases
Tsai, S. Q., & Joung, J. K. (2016) · Nature Reviews Genetics · doi:10.1038/nrg.2016.28 · Cited by 508 · see citing works
High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9Preprint
Varshney, G. K., Pei, W., LaFave, M. C., Idol, J., Xu, L., Gallardo, V. E., Carrington, B., Bishop, K. S., Jones, M. S., Li, M., et al. (2015) · Genome Research · doi:10.1101/gr.186379.114 · Cited by 510 · see citing works
“Three independent studies have shownCRISPR/Cas9 could inducemutations even at the sites with up to five mismatches (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”
BackgroundGonzãlez, F., Zhu, Z., Shi, Z., Lelli, K., Verma, N., Li, Q. V., & Huangfu, D. (2014) · Cell Stem Cell · doi:10.1016/j.stem.2014.05.018 · Cited by 486 · see citing works
“Recent studies suggest that the CRISPR/Cas system may have offtarget effects (Cho et al., 2014; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013), potentially confounding genetic studies in hPSCs.”
BackgroundIn vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice
Koblan, L. W., Erdos, M. R., Wilson, C., Cabral, W. A., Levy, J. M., Xiong, Z., Tavarez, U. L., Davison, L. M., Gete, Y. G., Mao, X., et al. (2021) · Nature · doi:10.1038/s41586-020-03086-7 · Cited by 479 · see citing works
Mapping the genomic landscape of CRISPR-Cas9 cleavage
Cameron, P., Fuller, C. K., Donohoue, P. D., Jones, B. N., Thompson, M. S., Carter, M. M., Gradia, S., Vidal, B., Garner, E., Slorach, E. M., et al. (2017) · Nature Methods · doi:10.1038/nmeth.4284 · Cited by 443 · see citing works
“Of note, the on-target site was recovered with the lowest concentration of nuclease that permitted recovery of target sites, most often at 0.25 nM sgRNP ( Supplementary Tables 2 – 9 ).”
BackgroundInfluentialVeres, A., Gosis, B. S., Ding, Q., Collins, R., Ragavendran, A., Brand, H., Erdin, S., Cowan, C. A., Talkowski, M. E., & Musunuru, K. (2014) · Cell Stem Cell · doi:10.1016/j.stem.2014.04.020 · Cited by 474 · see citing works
“…been demonstrated to produce off-target effects, i.e., mutagenesis at sites in the genome other than the desired on-target site (Hockemeyer et al., 2011; Mussolino et al., 2011; Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013; Cradick et al., 2013; Cho et al., 2014).”
InfluentialOusterout, D. G., Kabadi, A. M., Thakore, P. I., Majoros, W. H., Reddy, T. E., & Gersbach, C. A. (2015) · Nature Communications · doi:10.1038/ncomms7244 · Cited by 470 · see citing works
“This corroborates previous studies showing that lower levels of Cas9 and sgRNA can reduce off-target effects 14, 16 .”
Wang, X., Wang, Y., Wu, X., Wang, J., Wang, Y., Qiu, Z., Chang, T., Huang, H., Lin, R.-J., & Yee, J.-K. (2015) · Nature Biotechnology · doi:10.1038/nbt.3127 · Cited by 442 · see citing works
Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
Naeem, M., Majeed, S., Hoque, M. Z., & Ahmad, I. (2020) · Cells · doi:10.3390/cells9071608 · Cited by 457 · see citing works
Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems
Gomaa, A. A., Klumpe, H. E., Luo, M. L., Selle, K., Barrangou, R., & Beisel, C. L. (2014) · mBio · doi:10.1128/mbio.00928-13 · Cited by 437 · see citing works
DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing
Xue, C., & Greene, E. C. (2021) · Trends in Genetics · doi:10.1016/j.tig.2021.02.008 · Cited by 431 · see citing works
Mashiko, D., Fujihara, Y., Satouh, Y., Miyata, H., Isotani, A., & Ikawa, M. (2013) · Scientific Reports · doi:10.1038/srep03355 · Cited by 426 · see citing works
Rees, H. A., Komor, A. C., Yeh, W.-H., Caetano‐Lopes, J., Warman, M. L., Edge, A. S. B., & Liu, D. R. (2017) · Nature Communications · doi:10.1038/ncomms15790 · Cited by 422 · see citing works
Xie, S., Shen, B., Zhang, C., Huang, X., & Zhang, Y. (2014) · PLoS ONE · doi:10.1371/journal.pone.0100448 · Cited by 417 · see citing works
Small molecule–triggered Cas9 protein with improved genome-editing specificity
Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A., & Liu, D. R. (2015) · Nature Chemical Biology · doi:10.1038/nchembio.1793 · Cited by 418 · see citing works
Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae
Jakočiūnas, T., Bonde, I., Herrgård, M. J., Harrison, S. J., Kristensen, M., Pedersen, L. E., Jensen, M. K., & Keasling, J. D. (2015) · Metabolic Engineering · doi:10.1016/j.ymben.2015.01.008 · Cited by 412 · see citing works
Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities
van Dongen, J. E., Berendsen, J. T. W., Steenbergen, R. D. M., Wolthuis, R. M. F., Eijkel, J. C. T., & Segerink, L. I. (2020) · Biosensors and Bioelectronics · doi:10.1016/j.bios.2020.112445 · Cited by 420 · see citing works
“It was shown for Cas9 that less than 5% of crRNA sequences with two or more mismatches to the target sequence is functional in target binding (Anderson et al., 2015) and mismatches near the PAM region are less tolerated (Pattanayak et al., 2013).”
COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites
Cradick, T. J., Qiu, P., Lee, C. M., Fine, E. J., & Bao, G. (2014) · Molecular Therapy - Nucleic Acids · doi:10.1038/mtna.2014.64 · Cited by 408 · see citing works
Expanding the Biologist’s Toolkit with CRISPR-Cas9
Sternberg, S. H., & Doudna, J. A. (2015) · Molecular Cell · doi:10.1016/j.molcel.2015.02.032 · Cited by 398 · see citing works
“, 2013), leading many laboratories to analyze cleavage specificity more thoroughly using different approaches (Hsu et al., 2013; Mali et al., 2013a; Pattanayak et al., 2013).”
BackgroundUnraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach
Chari, R., Mali, P., Moosburner, M., & Church, G. M. (2015) · Nature Methods · doi:10.1038/nmeth.3473 · Cited by 394 · see citing works
Naturally Occurring Off-Switches for CRISPR-Cas9
Pawluk, A., Amrani, N., Zhang, Y., Garcı́a, B., Hidalgo-Reyes, Y., Lee, J., Edraki, A., Shah, M. K., Sontheimer, E. J., Maxwell, K. L., et al. (2016) · Cell · doi:10.1016/j.cell.2016.11.017 · Cited by 384 · see citing works
“For example, offtarget effects (cleavage and mutation at unintended, nearcognate genomic sites) are exacerbated by excessive or prolonged Cas9 activity (Fu et al., 2014; Hsu et al., 2013; Pattanayak et al., 2013).”
Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system
Sakuma, T., Nishikawa, A., Kume, S., Chayama, K., & Yamamoto, T. (2014) · Scientific Reports · doi:10.1038/srep05400 · Cited by 376 · see citing works
- 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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