The DOI matches its Crossref record; 39 of the 39 references it cites were found in a registry or an index.
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
Open accessAbstract
CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.
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“A primary limitation of GUIDE-seq is relevant to the low delivery efficiency of dsODNs into cells, which results in detection of only 30%–50% of all the DSBs (Tsai et al., 2015; Pan et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
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In the citing paper
“Off-target profiling of AsCas12a-crRNA+11 genome editing in HEK293/3272-26A>G cells18,42 showed very high specificity, as demonstrated by exclusive editing of the 3272-26A>G CFTR locus, while non-specific cleavages in the second allele, or any other genomic loci, could not be detected (Fig. 2c and Supplementary Fig. 4c).”
From Allele specific repair of splicing mutations in cystic fibrosis through AsCas12a genome editing (2019) · cites this paper as [42]
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In the citing paper
“The major barrier of clinical use of CRISPR/Cas9 is its off-target effects [81,82,83].”
From Viral Delivery Systems for CRISPR (2019) · cites this paper as [81]
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In the citing paper
“While cleavage at sites with perfect complementarity to the protospacer domain of the gRNA typically occurs with highest efficiency, cleavage at sites having one to several bases of mismatch with the gRNA can occur. These undesired cleavage events are called “off-target effects” (OTEs)10, and their existence should be minimized for therapeutic applications of genome editing technologies.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [10]
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“The GUIDE-seq procedure10 can be used to empirically identify real off-target sites in cells.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [10]
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“GUIDE-seq can detect off-target sites with indel frequencies as low as 0.03% (Tsai et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
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In the citing paper
“GUIDE-seq is more sensitive than the IDLV method because dsODNs integrate more efficiently and precisely into DSBs, while the integration events of IDLV is low in number and can distribute as far as 500bp away from the actual DSB sites (Tsai et al., 2015; Cromer et al., 2023).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
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“However, other publications observed a much lower validation rate (Cencic et al., 2014; Duan et al., 2014; Tsai et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
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“These studies indicated that Cas9 could associate with genomic loci without exerting its nuclease function, deposing a major caveat in Cas9 ChIP-Seq-based off-target detection assay.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
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“Interestingly, compared to cell-based methods such as GUIDE-seq (see next section) (Tsai et al., 2015), Extru-seq exhibited much lower false negative rate (2.3% versus 29%) (Kwon et al., 2023).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2015
In the citing paper
“Using the standard Guide-seq protocol, we detected only the Fah target site and one off-target site (defined as Guide-seq off-target site 1, or GOT1) (Supplementary Data Set 1).”
From Adenine base editing in an adult mouse model of tyrosinaemia (2019) · cites this paper as [23]
In the citing paper
“However, silicon methods can only identify part of the off-target cleavage.48”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [48]
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Citations
Cites
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Li, H., & Durbin, R. (2009) · Bioinformatics · doi:10.1093/bioinformatics/btp324 · Cited by 58,607
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
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Fast and accurate long-read alignment with Burrows-Wheeler transform.
Li, H., & Durbin, R. (2010) · Bioinformatics · doi:10.1093/bioinformatics/btp698 · Cited by 12,063
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
Development 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
DNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741
Double 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
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292
Self-citationCRISPR-Cas systems for editing, regulating and targeting genomes
Sander, J. D., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2842 · Cited by 3,153
Self-citationRNA-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
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Guide-specific loss of efficiency and off-target reduction with Cas9 variants
Zhang, L., He, W., Fu, R., Wang, S., Chen, Y., & Xu, H. (2023) · Nucleic Acids Research · doi:10.1093/nar/gkad702 · Cited by 27
Kwon, J., Kim, M., Hwang, W., Jo, A., Hwang, G.-H., Jung, M., Kim, U. G., Cui, G., Kim, H., Eom, J.-H., et al. (2023) · Genome biology · doi:10.1186/s13059-022-02842-4 · Cited by 24
Learning to quantify uncertainty in off-target activity for CRISPR guide RNAs
Özden, F., & Minary, P. (2024) · Nucleic Acids Research · doi:10.1093/nar/gkae759 · Cited by 21
Yaish, O., Asif, M., & Orenstein, Y. (2022) · Briefings in Bioinformatics · doi:10.1093/bib/bbac157 · Cited by 19
Hybrid multitask learning reveals sequence features driving specificity in the CRISPR/Cas9 system
Vora, D. S., Yadav, S., & Sundar, D. (2023) · Biomolecules · doi:10.3390/biom13040641 · Cited by 17
Genome-wide CRISPR off-target DNA break detection by the BLISS method
Ballarino, R., Bouwman, B. A. M., & Crosetto, N. (2021) · Methods in molecular biology · doi:10.1007/978-1-0716-0687-2_15 · Cited by 8
CRISPR off-target analysis platforms
Xu, C. L., Ruan, M. Z., Ragi, S. D., & Tsang, S. H. (2023) · Methods in molecular biology · doi:10.1007/978-1-0716-2651-1_26 · Cited by 6
New genetic modification techniques: challenges and prospects
Head, G., & Tzotzos, G. T. (2023) · Present Knowledge in Food Safety · doi:10.1016/b978-0-12-819470-6.00017-2 · 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
Self-citation
Metadata
- Authors
- Tsai, S. Q., Zheng, Z., Nguyen, N. T., Liebers, M., Topkar, V. V., Thapar, V., Wyvekens, N., Khayter, C., Iafrate, A. J., Le, L. P., Aryee, M. J., & Joung, J. K.
- Affiliations
- Harvard University (US)
- Massachusetts General Hospital (US)
- Corresponding author
- Tsai, Shengdar Q
- Joung, J Keith
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2014-12-16
- Volume
- 33
- Issue
- 2
- Pages
- 187-197
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4320685
- OpenAlex
- W2003797386
- Cited by
- 2,268
- References
- 39
- Citations by year
- 2026: 189 · 2025: 211 · 2024: 196 · 2023: 191 · 2022: 208 · 2021: 211 · 2020: 231 · 2019: 186 · 2018: 187 · 2017: 177 · 2016: 172 · 2015: 105 · 2014: 1
- FWCI
- 62.14
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Funders
- National Institute of General Medical Sciences (DP1GM105378, R01GM088040, F32GM105189)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR063070)
- National Center for Chronic Disease Prevention and Health Promotion (DP1 GM105378)
- 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
- GUIDE-seq, CRISPR-Cas nucleases, off-target cleavage, DNA double-strand breaks, genome-wide characterization, genome editing safety
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Cell Line, Humans, Oligodeoxyribonucleotides, Genome, Human, RNA Editing, DNA Breaks, Double-Stranded, High-Throughput Nucleotide Sequencing, CRISPR-Cas Systems
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