Cited by 1,487CRISPR and Genetic EngineeringRNA regulation and diseaseRNA Interference and Gene Delivery

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High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

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Pattanayak, Vikram, Lin, Steven, Guilinger, John P, Ma, Enbo, Doudna, Jennifer A, Liu, David R·2013·Nature Biotechnology

DOI 10.1038/nbt.2673PMC PMC3782611

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Abstract

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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Background
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Background
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“In addition, the dosage of the gRNA:Cas9 complex is very important for off‐target cleavage, as shown by more severe off‐ target effects at higher concentrations.(104,105) Collectively, these studies shed light on the rules of gRNA design, suggesting that judicious gRNA selection and experimental design could minimize…”
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Bailey, J. (2019) · BRILL eBooks · doi:10.1163/9789004391192_020 · Cited by 16
“Regardless of on-target efficiency, one issue has plagued the creation of GM animals: off-target effects, or mutations induced by the GM process that are not intended but affect other non-specific sites in the genome (Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013).”
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Designing Safer CRISPR/Cas9 Therapeutics for HIV: Defining Factors That Regulate and Technologies Used to Detect Off-Target Editing
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“In general, mismatches that were distal to PAM site had minimal effect on cleavage efficiency, which allowed CRISPR-mediated cleavage at off-target sites with similar, but not identical sequences (Fu et al., 2013; Hsu et al., 2013; Mali et al., 2013; Pattanayak et al., 2013).”
Background
Type II anti-CRISPR proteins as a new tool for synthetic biology
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“sgRNA design [27,79,104], rational engineering of Cas9 variants [105], and, lastly, the usage of anti-CRISPR proteins [93].”
Background
Eliminating predictable DNA off-target effects of cytosine base editor by using dual guiders including sgRNA and TALE
Zhou, J., Liu, Y., Wei, Y., Zheng, S., Gou, S., Chen, T., Yang, Y., Lan, T., Chen, M., Liao, Y. C., et al. (2022) · Molecular Therapy · doi:10.1016/j.ymthe.2022.04.010 · Cited by 14
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“(Lin et al. 2014), truncation of sgRNAs at the 5’ end or 3’ end (Pattanayak et al. 2013; Fu et al. 2014), addition of extra GG at the 5’ end of sgRNA, use of paired sgRNA Cas9 nickases; utilizing FokI nuclease are some of the reported strategies to reduce off-targeting and enhance target…”
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Chen, Y., Lü, W., Gao, N., Long, Y., Shao, Y., Liu, M., Chen, H., Ye, S., Ma, X., Li, D., et al. (2016) · Science China Life Sciences · doi:10.1007/s11427-016-5049-y · Cited by 15
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“The sequences of on-target and off-target sites differed by five nucleotides in other mammalian cells [14,26].”
Background
An efficient method to enrich for knock-out and knock-in cellular clones using the CRISPR/Cas9 system
Niccheri, F., Pecori, R., & Conticello, S. G. (2017) · Cellular and Molecular Life Sciences · doi:10.1007/s00018-017-2524-y · Cited by 15
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Generation of VDR Knock-Out Mice via Zygote Injection of CRISPR/Cas9 System
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Efficient Generation of Somatic Cell Nuclear Transfer-Competent Porcine Cells with Mutated Alleles at Multiple Target Loci by Using CRISPR/Cas9 Combined with Targeted Toxin-Based Selection System
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“It is known that CRISPR/Cas9-based genome editing often causes off-target mutations in irrelevant loci in certain cell types [35,36].”
Background
Efficient genome editing of genes involved in neural crest development using the CRISPR/Cas9 system in Xenopus embryos
Liu, Z., Cheng, T., Shi, Z., Liu, Z., Lei, Y., Wang, C., Shi, W., Chen, X., Qi, X., Cai, D., et al. (2016) · Cell & Bioscience · doi:10.1186/s13578-016-0088-4 · Cited by 14
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Background
Recent Biotechnological Advances in the Improvement of Cassava
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Background
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PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding
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“The repair mechanism of DSBs through error-prone non-homologous end joining (NHEJ), as described for human cells [48-51], seems to be absent in the malarial parasite.”
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Blighe, K., DeDionisio, L., Christie, K. A., Chawes, B., Shareef, S., Kakouli‐Duarte, T., Chao-Shern, C., Harding, V., Kelly, R. S., Castellano, L., et al. (2018) · BMC Genomics · doi:10.1186/s12864-018-4963-8 · Cited by 13
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Pannexin 1 Transgenic Mice: Human Diseases and Sleep-Wake Function Revision
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“It is known that CRISPR/Cas9 can have an off-target activity that can lead to unwanted mutations [37].”
Background
Natural Nucleoside Modifications in Guide RNAs Can Modulate the Activity of the CRISPR-Cas9 System In Vitro
Prokhorova, D. V., Vokhtantsev, I. P., Tolstova, P. O., Zhuravlev, E. S., Kulishova, L. M., Zharkov, D. O., & Stepanov, G. A. (2022) · The CRISPR Journal · doi:10.1089/crispr.2022.0069 · Cited by 13
GENOME EDITING FOR EARLY AND LATE FLOWERING IN PLANTS
Irfan, U., HAIDER, M., Shafiq, M., SAMI, A., & Ali, Q. (2023) · Bulletin of Biological and Allied Sciences Research · doi:10.54112/bbasr.v2023i1.45 · Cited by 14
“Strategies include choosing gRNA sequences with the fewest predicted off-target sites, optimizing gRNA length and composition, and considering secondary structure predictions (Pattanayak et al., 2013).”
TALEN‐mediated gene editing of the thrombospondin‐1 locus in axolotl
Kuo, T., Kowalko, J. E., DiTommaso, T., Nyambi, M., Montoro, D. T., Essner, J. J., & Whited, J. L. (2015) · Regeneration · doi:10.1002/reg2.29 · Cited by 13
Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)‐associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development
Cheng, M., Jin, X., Mu, L., Wang, F., Li, W., Zhong, X., Liu, X., Shen, W., Liu, Y., & Zhou, Y. (2016) · Journal of Neuroscience Research · doi:10.1002/jnr.23776 · Cited by 13
“, 2015) and off-target effects (Fu et al., 2013; Pattanayak et al., 2013; Cho et al., 2014; Kuscu et al., 2014) must be further optimized and overcome (Ran et al.”
BackgroundInfluential
Recent advances in DNA-free editing and precise base editing in plants
Zhang, Y., & Gao, C. (2017) · Emerging Topics in Life Sciences · doi:10.1042/etls20170021 · Cited by 16
Gene editing as a promising approach for respiratory diseases
Bai, Y., Liu, Y., Su, Z., Ma, Y., Ren, C., Zhao, R., & Ji, H. (2018) · Journal of Medical Genetics · doi:10.1136/jmedgenet-2017-104960 · Cited by 15
Anticipating emerging biotechnology threats
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Divalent cations promote TALE DNA-binding specificity
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Genome editing in the nematode Caenorhabditis briggsae using the CRISPR/Cas9 system
Culp, E. J., Richman, C. M., Sharanya, D., Jhaveri, N. S., van den Berg, W., & Gupta, B. P. (2020) · Biology Methods and Protocols · doi:10.1093/biomethods/bpaa003 · Cited by 14
T-complex protein 1 subunit zeta-2 (CCT6B) deficiency induces murine teratospermia
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Screening DNA aptamers that control the DNA cleavage, homology-directed repair, and transcriptional regulation of the CRISPR-(d)Cas9 system
Huang, X., Wang, M., Wu, X., Zou, Y., Xu, J., Cao, C., Ma, Q., Yu, B., Liu, Y., & Gui, Y. (2022) · Molecular Therapy · doi:10.1016/j.ymthe.2022.10.009 · Cited by 13
CRISPR/Cas9-mediated Targeted Mutagenesis of Inulin Biosynthesis in Rubber Dandelion
Ariyaratne, M., King-Smith, N. P., Fresnedo‐Ramírez, J., Barker, D. J., & Cornish, K. (2023) · Journal of the American Society for Horticultural Science · doi:10.21273/jashs05311-23 · Cited by 12
An engineered baculoviral protein and DNA co-delivery system for CRISPR-based mammalian genome editing
Capin, J., Harrison, A., Raele-Rolfe, R., Yadav, S. K. N., Baiwir, D., Mazzucchelli, G., Quinton, L., Satchwell, T. J., Toye, A. M., Schaffitzel, C., et al. (2024) · Nucleic Acids Research · doi:10.1093/nar/gkae142 · Cited by 12
“Moreover, their propensity for prolonged transgene expression in target cells increases the risk of undesired and often detrimental off-target effects ( 10–12 ).”
Background
OliTag-seq enhances in cellulo detection of CRISPR-Cas9 off-targets
Yang, Z.-X., Deng, D.-H., Gao, Z.-Y., Zhang, Z.-K., Fu, Y.-W., Wen, W., Zhang, F., Li, X., Li, H., Zhang, J., et al. (2024) · Communications Biology · doi:10.1038/s42003-024-06360-w · Cited by 17
Application of CRISPR Technology in Plant Improvement: An Update Review
Saini, H., Devrani, A., Synrem, G. J., & Priyanka (2025) · Advances in Agriculture · doi:10.1155/aia/4578877 · Cited by 15
Machine learning and deep learning for genomic data: a data-centric approach to CRISPR/Cas9 gene editing
Rafiq, S., Macha, M. A., & Assad, A. (2025) · International Journal of Data Science and Analytics · doi:10.1007/s41060-025-00773-4 · Cited by 13
In vivo delivery systems for CRISPR genome editing: Viral and non-viral carriers
Lee, Y., Kim, B., Lee, D., Cheon, S. Y., Lim, S. G., Kim, Y., & Koo, H. (2025) · Applied Physics Reviews · doi:10.1063/5.0228874 · Cited by 13
Massively parallel determination and modeling of endonuclease substrate specificity
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“(24), but they have so far only been tested on a small scale.”
Background
Bioinformatics analyses of Shigella CRISPR structure and spacer classification
Wang, P., Zhang, B., Duan, G., Wang, Y., Hong, L., Wang, L., Guo, X., Xi, Y., & Yang, H. (2016) · World Journal of Microbiology and Biotechnology · doi:10.1007/s11274-015-2002-3 · Cited by 12
“The key event is the speciﬁc combination between CRISPR/Cas9 and the target sites (Kuscu et al. 2014; Pattanayak et al. 2013).”
Background
Nucleases in gene-editing technologies: past and prologue
Li, D., Li, L.-Q., & Liu, J. (2023) · National Science Open · doi:10.1360/nso/20220067 · Cited by 12
“Previous studies on the off-target cleavage of guide RNA: Cas9 complex suggested that using a shorter less-active guide RNA or decreasing the RNP concentration would limit the enzyme activity and lead to higher target specificity [153].”
Background
The energy landscape for R-loop formation by the CRISPR–Cas Cascade complex
Kauert, D. J., Madariaga-Marcos, J., Rutkauskas, M., Wulfken, A., Songailienė, I., Šinkūnas, T., Šikšnys, V., & Seidel, R. (2023) · Nature Structural & Molecular Biology · doi:10.1038/s41594-023-01019-2 · Cited by 12
Efficient gene-targeting in rat embryonic stem cells by CRISPR/Cas and generation of human kynurenine aminotransferase II (KAT II) knock-in rat
Yamamoto, S., Ooshima, Y., Nakata, M., Yano, T., Nishimura, N., Nishigaki, R., Satomi, Y., Matsumoto, H., Matsumoto, Y., & Takeyama, M. (2015) · Transgenic Research · doi:10.1007/s11248-015-9909-1 · Cited by 12
Generation of an Oocyte-Specific Cas9 Transgenic Mouse for Genome Editing
Zhang, L.-L., Zhou, J., Han, J., Hu, B., Hou, N., Shi, Y. S., Huang, X., & Lou, X. (2016) · PLoS ONE · doi:10.1371/journal.pone.0154364 · Cited by 12
Targeting Human α-Lactalbumin Gene Insertion into the Goat β-Lactoglobulin Locus by TALEN-Mediated Homologous Recombination
Zhu, H., Liu, J., Cui, C., Song, Y., Ge, H., Hu, L., Li, Q., Jin, Y., & Zhang, Y. (2016) · PLoS ONE · doi:10.1371/journal.pone.0156636 · Cited by 12
“For RGEN technique, a rising number of researches have revealed high potential off-target cleavage due to three or fewer mismatches in their guide RNAs and the difficulty of controlling the concentrations of Cas9:guide RNA complexes [31, 32].”
Background
CRISPcut: A novel tool for designing optimal sgRNAs for CRISPR/Cas9 based experiments in human cells
Dhanjal, J. K., Radhakrishnan, N., & Sundar, D. (2018) · Genomics · doi:10.1016/j.ygeno.2018.03.011 · Cited by 12
The Potential Use of the CRISPR-Cas System for HIV-1 Gene Therapy
Sanches-da-Silva, G. D. N., Medeiros, L. F. S., & Lima, F. M. (2019) · International Journal of Genomics · doi:10.1155/2019/8458263 · Cited by 13
“Pioneering studies using paired Cas9 nickases found extensive reduction of off-target events [83, 84] and did not find chromosomal translocations [85].”
Comprehensive Analysis of CRISPR/Cas9-Mediated Mutagenesis in Arabidopsis thaliana by Genome-Wide Sequencing
Xu, W., Fu, W., Zhu, P., Li, Z., Wang, C., Wang, C., Zhang, Y., & Zhu, S. (2019) · International Journal of Molecular Sciences · doi:10.3390/ijms20174125 · Cited by 13
“suggested that perfect complementarity between sgRNA and target sequences is required in the 7–12 base pairs adjacent to the PAM [47].”
Background
The CRISPR-Cas system – from bacterial immunity to genome engineering
Czarnek, M., & Bereta, J. (2016) · Postępy Higieny i Medycyny Doświadczalnej · doi:10.5604/17322693.1216379 · Cited by 13
Tandem repeat knockout utilizing the CRISPR/Cas9 system in human cells
Lv, Q., Lai, L., Yuan, L., Song, Y., Sui, T., & Li, Z. (2016) · Gene · doi:10.1016/j.gene.2016.02.013 · Cited by 11
Challenges and opportunities when transitioning from in vivo gene replacement to in vivo CRISPR/Cas9 therapies – a spotlight on hemophilia
Segurado, O. G., Jiang, R., & Pipe, S. W. (2022) · Expert Opinion on Biological Therapy · doi:10.1080/14712598.2022.2090241 · Cited by 11
Efficient Cas9‐based Genome Editing Using CRISPR Analysis Webtools in Severe Early‐onset‐obesity Patient‐derived iPSCs
Patel, A., Iannello, G., Diaz, A. G., Sirabella, D., Thaker, V., & Corneo, B. (2022) · Current Protocols · doi:10.1002/cpz1.519 · Cited by 11
“It has been reported, however, that excessive or limited Cas9 or sgRNA can undermine the specificity and efficiency of targeting (Hsu et al., 2013; Pattanayak et al., 2013; Wu et al., 2014).”
Background
Application of CRISPR for In Vivo Mouse Cancer Studies
Thomsen, M. K. (2022) · Cancers · doi:10.3390/cancers14205014 · Cited by 11
“have been raised, as guides are kept being expressed in the cells together with Cas9 [9,31].”
Background
Recent developments, challenges and opportunities in genome editing for crop science from a societal perspective
Venkataraman, S., Zaruk, D., & Hefferon, K. L. (2025) · Frontiers in Genome Editing · doi:10.3389/fgeed.2025.1568072 · Cited by 13
Multi-OMICs and Genome Editing Perspectives on Liver Cancer Signaling Networks
Lin, S., Yin, Y. A., Jiang, X., Sahni, N., & Yi, S. S. (2016) · BioMed Research International · doi:10.1155/2016/6186281 · Cited by 12
“The seed sequence close to the PAM domain carries more weight in target specificity, while the mismatches further away from the PAM domain and towards the 5-end of the targeted genome sequence could be tolerated to certain degree [145–147].”
Background
The present and future of genome editing in cancer research
Li, X., Wu, R., & Ventura, A. (2016) · Human Genetics · doi:10.1007/s00439-016-1713-3 · Cited by 12
“Other than the dangers associated with random integration of lentiviral and retroviral vectors, undesired off-target effects of CRISPR need to be considered carefully before moving to the clinic (Pattanayak et al. 2013).”
Sequencing data from Massachusetts General Hospital shows Cas9 integration into the genome, highlighting a serious hazard in gene-editing therapeutics
Chakraborty, S. (2019) · F1000Research · doi:10.12688/f1000research.20744.1 · Cited by 12
Enhanced genome editing in human iPSCs with CRISPR-CAS9 by co-targeting ATP1a1
Liu, J.-T., Corbett, J., Heslop, J. A., & Duncan, S. A. (2020) · PeerJ · doi:10.7717/peerj.9060 · Cited by 11
Unexpected gene activation following CRISPR‐Cas9‐mediated genome editing
Manjón, A. G., Linder, S., Teunissen, H., Friskes, A., Zwart, W. T., de Wit, E., & Medema, R. H. (2021) · EMBO Reports · doi:10.15252/embr.202153902 · Cited by 10

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
ORCID: Pattanayak, Vikram
Lin, Steven
Doudna, Jennifer A
Liu, David R
Venue: Nature Biotechnology
Publisher: Springer Science and Business Media LLC
Published: 2013-08-11
Volume: 31
Issue: 9
Pages: 839-843
ISSN: 0733-222X (ISSN-L)
1087-0156
1546-1696
DOI: 10.1038/nbt.2673
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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High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity

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