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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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“Recent large-scale systematic reports revealed an unexpectedly high frequency off-target effects using the CRISPR/Cas system in several human cell lines (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013).”
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Efficient CRISPR/Cas9-Mediated Gene Knockin in Mouse Hematopoietic Stem and Progenitor Cells
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Specificity profiling of CRISPR system reveals greatly enhanced off-target gene editing
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“Cleavage and mutation at unexpected sites or homologous genomic sites might interfere with therapeutic analysis or harm an organism via excessive or prolonged Cas9 activity (10).”
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Luhur, A., Klueg, K. M., & Zelhof, A. C. (2018) · Wiley Interdisciplinary Reviews Developmental Biology · doi:10.1002/wdev.339 · Cited by 31
“1 | sgRNA design, off target effects, and validation One of the challenges associated with CRISPR-based gene disruption for functional genomics pertain to both sgRNA design and the underexplored OTEs of the gRNAs (Fu et al., 2013; Pattanayak et al., 2013).”
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CRISPR‐Mediated Gene Targeting of Human Induced Pluripotent Stem Cells
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“…a population of immortalized tumor cell lines found substantial rates of off-target Cas9 nuclease activity (Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013; Kuscu et al., 2014), this may be less of a concern for gene editing individual stem cell clones (Wu et al., 2014; Yang et…”
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InfluentialCharacterizing Protease Specificity: How Many Substrates Do We Need?
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Genome Engineering with TALE and CRISPR Systems in Neuroscience
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“Off-target mutation frequency can be high when using CRISPR/Cas9 technology (Pattanayak et al. 2013) because Cas9 with sgRNA has potential off-target cleavage activity on DNA sequences even with three to five base pair mismatches in the PAM-distal part of sgRNA sequence (Fu et al.”
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“Mismatches within the guide-target heteroduplex are nevertheless tolerated in some positions, and this is the chief source of off-target activities in Cas9-based gene targeting applications (Fu et al., 2013; Hsu et al., 2013; Mali, Aach, et al., 2013; Pattanayak et al., 2013).”
BackgroundGeneration of genetically-engineered animals using engineered endonucleases
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“The off-target issue is also the biggest concern in the CRISPR/Cas9 system (Fu et al. 2013; Hsu et al. 2013; Mali et al. 2013a; Pattanayak et al. 2013) and various efforts have been made to improve its specificity.”
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Li, M., Chen, F., Yang, Q., Tang, Q., Xiao, Z., Tong, X., Zhang, Y., Lei, L., & Li, S. (2024) · Biomaterials Research · doi:10.34133/bmr.0023 · Cited by 32
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The zebrafish genome editing toolkit
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“Upon emergence of the CRISPR/Cas9 methodology, it was soon realized that minimizing CRISPR/Cas9 offtarget effects was required (Fu et al., 2013; Hsu et al., 2013; Jinek et al., 2012; Pattanayak et al., 2013).”
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“Depending on the sequence of exact on-target site, CRISPR-Cas9 may edit a large number of unintended sites (Cradick et al., 2013; Fu et al., 2013; Pattanayak et al., 2013; Tsai et al., 2015).”
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Sivakumar, A., & Cherqui, S. (2022) · Frontiers in Genome Editing · doi:10.3389/fgeed.2022.903139 · Cited by 32
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“The high frequency of off-target activity in a genome-wide scale associated with CRISPR/Cas9 is still one major concern impeding biomedical and clinical application (Fu et al., 2013; Kuscu et al., 2014; Pattanayak et al., 2013; Wu et al., 2014).”
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BackgroundCRISPR Diagnosis and Therapeutics with Single Base Pair Precision
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“, 5′-NAG) has also been reported, albeit at less efficient rates (Jinek et al., 2012; Jiang et al., 2013; Pattanayak et al., 2013).”
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“The off-target alterations identified in these investigations harbored up to five nucleotide substitutions between the hybridizing sgRNA and the target genomic sequence [42,46], raising concerns with respect to therapeutic applications as frequent sequences with five mismatches to a 20-nucleotide sgRNA may be found in…”
BackgroundGeneration of knockout mice using engineered nucleases
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TRANSCRIPTION FACTOR Bmsage PLAYS A CRUCIAL ROLE IN SILK GLAND GENERATION IN SILKWORM, Bombyx mori
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“It has been reported that PAM type and mismatch site from the seed region or nonseed region have an influence on the specificity of the CRISPR/Cas9 system (Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013).”
BackgroundCRISPR-Cas9: From a bacterial immune system to genome-edited human cells in clinical trials
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“The major challenge of the CRISPR/Cas system is the off-target mutation and editing efficiency (Fu et al. 2013; Pattanayak et al. 2013).”
BackgroundAllosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides
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“The aforementioned in vitro and in vivo data have suggested that the inhibitory effects of G8P on CRISPR-Cas9 is dependent on the accessibility of Cas9 protein to sgRNA.”
BackgroundInfluentialOptical Manipulation of CRISPR/Cas9 Functions: From Ultraviolet to Near-Infrared Light
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Combining CRISPR/Cas9 and rAAV Templates for Efficient Gene Editing
Kaulich, M., & Dowdy, S. F. (2015) · Nucleic Acid Therapeutics · doi:10.1089/nat.2015.0545 · Cited by 25
“However, several open questions remain: (1) How to identify and validate off-target effects? (2) How to avoid the CRISPR/ Cas9-mediated cut on the second strand for heterozygous targeting strategies? (3) How to increase the overall targeting efficiency with HDR templates that do not contain selectable markers? First,…”
MethodEfficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein
Jung, C., Zhang, J., Trenchard, E., Lloyd, K. C. K., West, D. B., Rosen, B. P., & de Jong, P. J. (2016) · Transgenic Research · doi:10.1007/s11248-016-9998-5 · Cited by 26
“…we explored the option of using the obligate dimeric properties of dCas9–FokI to strengthen sgRNA specificity, because some reports raised concerns about high off-target mutation rates induced by CRISPR/Cas9 in human cells (Fu et al. 2013; Hsu et al. 2013; Pattanayak et al. 2013; Lin et al. 2014).”
BackgroundHu, W., Kumar, A., Ahmed, S. F., Qi, S., Ma, D. K. G., Chen, H., Singh, G. J., Casan, J. M. L., Haber, M., Voskoboinik, I., et al. (2024) · Nature Structural & Molecular Biology · doi:10.1038/s41594-024-01336-0 · Cited by 24
The potential of HBV cure: an overview of CRISPR-mediated HBV gene disruption
Yao, Z. Q., Schank, M., Zhao, J., Gazzar, M. E., Wang, L., Zhang, Y., Hill, A. C., Banik, P., Pyburn, J. S., & Moorman, J. P. (2024) · Frontiers in Genome Editing · doi:10.3389/fgeed.2024.1467449 · Cited by 28
Finnigan, G. C., & Thorner, J. W. (2016) · G3 Genes Genomes Genetics · doi:10.1534/g3.116.029801 · Cited by 26
CRISPR/Cas9 in the era of nanomedicine and synthetic biology
Schmidt, T. J. N., Berarducci, B., Konstantinidou, S., & Raffa, V. (2022) · Drug Discovery Today · doi:10.1016/j.drudis.2022.103375 · Cited by 26
Carnielli, J. B. T., Dave, A., Romano, A., Forrester, S. J., de Faria, P. R., Monti-Rocha, R., Costa, C. H. N., Dietze, R., Graham, I. A., & Mottram, J. C. (2022) · EBioMedicine · doi:10.1016/j.ebiom.2022.104378 · Cited by 25
Generating Genetically Modified Mice: A Decision Guide
Huijbers, I. J. (2017) · Methods in molecular biology · doi:10.1007/978-1-4939-7169-5_1 · Cited by 23
Efficient SSA ‐mediated precise genome editing using CRISPR /Cas9
Li, X., Bai, Y., Cheng, X., Kalds, P., Sun, B., Wu, Y., Lv, H., Xu, K., & Zhang, Z. (2018) · FEBS Journal · doi:10.1111/febs.14626 · Cited by 23
Advances in therapeutic application of CRISPR-Cas9
Sun, J., Wang, J., Zheng, D., & Hu, X. (2019) · Briefings in Functional Genomics · doi:10.1093/bfgp/elz031 · Cited by 25
Lipid and polymer mediated CRISPR/Cas9 gene editing
Gong, Y., Tian, S., Yang, X., & Zhang, S. (2020) · Journal of Materials Chemistry B · doi:10.1039/d0tb00207k · Cited by 25
Exploring the potential of cell-derived vesicles for transient delivery of gene editing payloads
Leandro, K., Rufino‐Ramos, D., Breyne, K., Ianni, E. D., Lopes, S., Nobre, R. J., Kleinstiver, B. P., Perdigão, P. R. L., Breakefield, X. O., & de Almeida, L. P. (2024) · Advanced Drug Delivery Reviews · doi:10.1016/j.addr.2024.115346 · Cited by 27
Prime Editing: A Revolutionary Technology for Precise Treatment of Genetic Disorders
Li, M., Lin, Y., Cheng, Q., & Wei, T. (2025) · Cell Proliferation · doi:10.1111/cpr.13808 · Cited by 28
“Besides, DSBs may result in substantial translocations, rearrangements, activation of the p53 gene, and other genomic damages, largely limiting the clinical application of the CRISPR/Cas9 system [17–19].”
The multiplexed CRISPR targeting platforms
Cao, J. P., Xiao, Q., & Yan, Q. (2018) · Drug Discovery Today Technologies · doi:10.1016/j.ddtec.2018.01.001 · Cited by 27
Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity
Seamon, K. J., Light, Y. K., Saada, E. A., Schoeniger, J. S., & Harmon, B. (2018) · Analytical Chemistry · doi:10.1021/acs.analchem.8b01155 · Cited by 24
Moir-Meyer, G., Cheong, P. L., Olijnik, A.-A., Brown, J. M., Knight, S. J. L., King, A. J., Kurita, R., Nakamura, Y., Gibbons, R. J., Higgs, D. R., et al. (2018) · Methods and Protocols · doi:10.3390/mps1030028 · Cited by 24
“Off-target sites can differ from the targeted locus by up to seven nucleotides [39,42] and these can be in the form of single nucleotide mismatches, small indels or DNA-RNA bulges [33].”
BackgroundMurugan, K., Suresh, S. K., Seetharam, A. S., Severin, A. J., & Sashital, D. G. (2021) · Nucleic Acids Research · doi:10.1093/nar/gkab163 · Cited by 25
“The higher tolerance for a single mismatch in the seed sequence is likely due to the relatively high concentration of Cas9 used for pLibrary cleavage (18).”
InfluentialLiu, X., Yang, J., Song, Y., Zhang, X., Wang, X., & Wang, Z. (2021) · The Crop Journal · doi:10.1016/j.cj.2021.05.015 · Cited by 24
Current AAV-mediated gene therapy in sensorineural hearing loss
Qi, J., Fu, X., Zhang, L., Tan, F., Li, N., Sun, Q., Hu, X., He, Z., Xia, M., & Chai, R. (2022) · Fundamental Research · doi:10.1016/j.fmre.2022.08.015 · Cited by 24
Tripathi, S., Khatri, P., Fatima, Z., Pandey, R. P., & Hameed, S. (2022) · Pathogens · doi:10.3390/pathogens12010056 · Cited by 27
“Despite this, nucleic acid editing techniques have the potential to cause desired alterations on the target DNA [14,134].”
BackgroundRossi, M., Steklov, M. Y., Huberty, F., Nguyen, T., Marijsse, J., Jacques-Hespel, C., Najm, P., Lonez, C., & Breman, E. (2023) · Molecular Therapy — Nucleic Acids · doi:10.1016/j.omtn.2023.102038 · Cited by 26
Zhang, Y., Arango, G., Li, F., Xiao, X., Putatunda, R., Yu, J., Yang, X. F., Wang, H., Watson, L. T., Zhang, L., et al. (2018) · BMC Medical Genomics · doi:10.1186/s12920-018-0394-2 · Cited by 25
Carpenter, M. A., Law, E. K., Serebrenik, A. A., Brown, W. L., & Harris, R. S. (2018) · Methods · doi:10.1016/j.ymeth.2018.12.006 · Cited by 24
Engineering of Cas12a nuclease variants with enhanced genome-editing specificity
Chen, P., Zhou, J., Liu, H., Zhou, E.-C., He, B., Wu, Y., Wang, H., Sun, Z., Paek, C., Lei, J., et al. (2024) · PLoS Biology · doi:10.1371/journal.pbio.3002514 · Cited by 28
Strategies for improving the genome-editing efficiency of class 2 CRISPR/Cas system
Wang, L., & Han, H. (2024) · Heliyon · doi:10.1016/j.heliyon.2024.e38588 · Cited by 26
Saeed, K., Ayub, F., Durrani, M. A., & Mujahid, M. (2025) · The Microbe · doi:10.1016/j.microb.2025.100344 · Cited by 26
Utilization of TALEN and CRISPR/Cas9 technologies for gene targeting and modification
Pu, J., Frescas, D., Zhang, B., & Feng, J. (2015) · Experimental Biology and Medicine · doi:10.1177/1535370215584932 · Cited by 23
Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans
Bell, R. T., Fu, B. X. H., & Fire, A. Z. (2015) · Genetics · doi:10.1534/genetics.115.185041 · Cited by 23
“These results are consistent with previous observations of strong but not perfect PAM dependence for both wild-type Cas9 (Hsu et al. 2013; Pattanayak et al. 2013; Fu et al. 2014; Wu et al. 2014) and the VQR/VRER variants (Kleinstiver et al. 2015).”
ResultIdentification of a Xist silencing domain by Tiling CRISPR
Wang, Y., Yang, Z. H., Zhou, Y., Tanaseichuk, O., Li, Z., & Zhao, J. C. (2019) · Scientific Reports · doi:10.1038/s41598-018-36750-0 · Cited by 23
The CRISPR/Cas9 induces large genomic fragment deletions of MSTN and phenotypic changes in sheep
Ding, Y., Zhou, S., Ding, Q., Bei, C. A. I., Zhao, X., Zhong, S., Jin, M., Wang, X., Ma, B., & Chen, Y. (2020) · Journal of Integrative Agriculture · doi:10.1016/s2095-3119(19)62853-4 · Cited by 23
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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