Genome-wide CRISPR off-target DNA break detection by the BLISS method
Abstract
Clustered regularly interspaced palindromic repeat (CRISPR) systems are revolutionizing many areas of biology and medicine, where they are increasingly utilized as therapeutic tools for correcting disease-causing mutations. From a clinical perspective, unintended off-target (OT) DNA double-strand break (DSB) induction by CRISPR nucleases represents a major concern. Therefore, in recent years considerable effort has been dedicated to developing methods for assessing the OT activity of CRISPR nucleases, which in turn can be used to guide engineering of nucleases with minimal OT activity. Here we describe a detailed protocol for quantifying OT DSBs genome-wide in cultured cells transfected with CRISPR enzymes, based on the breaks labeling in situ and sequencing (BLISS) method that we have previously developed. CRISPR-BLISS is versatile and scalable, and allows assessment of multiple guide RNAs in different cell types and time points following cell transfection or transduction.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
BLISS ligates DSB ends with adapters containing the T7 promoter, so tagged DNA fragments can be linearly amplified via T7-mediated transcription before sequencing.
“BLISS ligates DSB ends with adapters containing the T7 promoter, so the tagged DNA fragments can be linearly amplified via T7-mediated transcription before sequencing (Yan et al., 2017; Ballarino et al., 2021).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Ballarino et al., 2021
Cites
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 · see citing works
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 · see citing works
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
Gilbert, L. A., Horlbeck, M. A., Adamson, B., Villalta, J. E., Chen, Y., Whitehead, E. H., Guimaraes, C., Panning, B., Ploegh, H. L., Bassik, M. C., et al. (2014) · Cell · doi:10.1016/j.cell.2014.09.029 · Cited by 2,728 · see citing works
In vivo genome editing using Staphylococcus aureus Cas9
Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., et al. (2015) · Nature · doi:10.1038/nature14299 · Cited by 2,687 · see citing works
High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects
Kleinstiver, B. P., Pattanayak, V., Prew, M. S., Tsai, S. Q., Nguyen, N. T., Zheng, Z., & Joung, J. K. (2016) · Nature · doi:10.1038/nature16526 · Cited by 2,673 · see citing works
Rationally engineered Cas9 nucleases with improved specificity
Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., & Zhang, F. (2016) · Science · doi:10.1126/science.aad5227 · Cited by 2,414 · see citing works
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
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., et al. (2015) · Nature Biotechnology · doi:10.1038/nbt.3117 · Cited by 2,268 · see citing works
Kosicki, M., Tomberg, K., & Bradley, A. (2018) · Nature Biotechnology · doi:10.1038/nbt.4192 · Cited by 1,883 · see citing works
A Protein-Tagging System for Signal Amplification in Gene Expression and Fluorescence Imaging
Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S., & Vale, R. D. (2014) · Cell · doi:10.1016/j.cell.2014.09.039 · Cited by 1,559 · see citing works
Negative magnetoresistance without well-defined chirality in the Weyl semimetal TaP
Arnold, F., Shekhar, C., Wu, S.-C., Sun, Y., dos Reis, R. D., Kumar, N., Naumann, M., Ajeesh, M. O., Schmidt, M. P., Grushin, A. G., et al. (2016) · Nature Communications · doi:10.1038/ncomms11615 · Cited by 1,341 · see citing works
Thakore, P. I., D'Ippolito, A. M., Song, L., Safi, A., Shivakumar, N. K., Kabadi, A. M., Reddy, T. E., Crawford, G. E., & Gersbach, C. A. (2015) · Nature Methods · doi:10.1038/nmeth.3630 · Cited by 1,012 · see citing works
Digenome-seq: Genome-wide profiling of CRISPR-cas9 off-target effects in human cells
Kim, D., Bae, S., Park, J., Kim, E., Kim, S., Yu, H. R., Hwang, J., Kim, J.-I., & Kim, J.-S. (2015) · Nature Methods · doi:10.1038/nmeth.3284 · Cited by 992 · see citing works
Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9
Paquet, D., Kwart, D., Chen, A., Sproul, A., Jacob, S., Teo, S., Olsen, K. M., Gregg, A., Noggle, S., & Tessier-Lavigne, M. (2016) · Nature · doi:10.1038/nature17664 · Cited by 951 · see citing works
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
Wu, X., Scott, D. A., Kriz, A. J., Chiu, A. C., Hsu, P. D., Dadon, D. B., Cheng, A. W., Trevino, A. E., Konermann, S., Chen, S., et al. (2014) · Nature Biotechnology · doi:10.1038/nbt.2889 · Cited by 944 · see citing works
CIRCLE-Seq: A highly sensitive in vitro screen for genome-wide CRISPR-cas9 nuclease off-targets
Tsai, S. Q., Nguyen, N. T., Malagon-Lopez, J., Topkar, V. V., Aryee, M. J., & Joung, J. K. (2017) · Nature Methods · doi:10.1038/nmeth.4278 · Cited by 814 · see citing works
Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease
Kuscu, C., Arslan, S., Singh, R., Thorpe, J., & Adli, M. (2014) · Nature Biotechnology · doi:10.1038/nbt.2916 · Cited by 777 · see citing works
Cheng, A. W., Wang, H., Yang, H., Shi, L., Yarden Katz, A., Theunissen, T. W., Rangarajan, S., Shivalila, C., Dadon, D. B., & Jaenisch, R. (2013) · Cell Research · doi:10.1038/cr.2013.122 · Cited by 743 · 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
Lin, 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
Rock, J. M., Hopkins, F. F., Chavez, A., Diallo, M., Chase, M. R., Gerrick, E. R., Pritchard, J. R., Church, G. M., Rubin, E. J., Sassetti, C. M., et al. (2017) · Nature Microbiology · doi:10.1038/nmicrobiol.2016.274 · Cited by 538 · see citing works
Chiarle, R., Zhang, Y., Frock, R. L., Lewis, S. M., Molinié, B., Ho, Y.-J., Myers, D. R., Choi, V. W., Compagno, M., Malkin, D. J., et al. (2011) · Cell · doi:10.1016/j.cell.2011.07.049 · Cited by 475 · see citing works
Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing
Crosetto, N., Mitra, A., Silva, M. J., Bienko, M., Dojer, N., Wang, Q., Karaca, E., Chiarle, R., Skrzypczak, M., Ginalski, K., et al. (2013) · Nature Methods · doi:10.1038/nmeth.2408 · Cited by 465 · see citing works
Self-citationEngineered Cpf1 variants with altered PAM specificities
Gao, L., Cox, D. B. T., Yan, W. X., Manteiga, J. C., Schneider, M. W., Yamano, T., Nishimasu, H., Nureki, O., Crosetto, N., & Zhang, F. (2017) · Nature Biotechnology · doi:10.1038/nbt.3900 · Cited by 460 · see citing works
Self-citationMapping 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
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
Genome Organization Drives Chromosome Fragility
Canela, A., Maman, Y., Jung, S., Wong, N., Callen, E., Day, A., Kieffer-Kwon, K.-R., Pekowska, A., Zhang, H., Rao, S. S. P., et al. (2017) · Cell · doi:10.1016/j.cell.2017.06.034 · Cited by 383 · see citing works
BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks
Yan, W. X., Mirzazadeh, R., Garnerone, S., Scott, D., Schneider, M. W., Kallas, T., Custodio, J., Wernersson, E., Li, Y., Gao, L., et al. (2017) · Nature Communications · doi:10.1038/ncomms15058 · Cited by 369 · see citing works
Self-citationDynamics of CRISPR-Cas9 genome interrogation in living cells
Knight, S. C., Xie, L., Deng, W., Guglielmi, B., Witkowsky, L. B., Bosanac, L., Zhang, E., Beheiry, M. E., Masson, J., Dahan, M., et al. (2015) · Science · doi:10.1126/science.aac6572 · Cited by 350 · see citing works
In vivo CRISPR editing with no detectable genome-wide off-target mutations
Akçakaya, P., Bobbin, M. L., Guo, J. A., Malagon-Lopez, J., Clement, K., Garcia, S. P., Fellows, M. D., Porritt, M. J., Firth, M. A., Carreras, A., et al. (2018) · Nature · doi:10.1038/s41586-018-0500-9 · Cited by 316 · see citing works
DNA Breaks and End Resection Measured Genome-wide by End Sequencing
Canela, A., Sridharan, S., Sciascia, N., Tubbs, A., Meltzer, P., Sleckman, B. P., & Nussenzweig, A. (2016) · Molecular Cell · doi:10.1016/j.molcel.2016.06.034 · Cited by 284 · see citing works
Activities and specificities of CRISPR /Cas9 and Cas12a nucleases for targeted mutagenesis in maize
Lee, K., Zhang, Y., Kleinstiver, B. P., Guo, J. A., Aryee, M. J., Miller, J., Malzahn, A. A., Zarecor, S., Lawrence‐Dill, C. J., Joung, J. K., et al. (2018) · Plant Biotechnology Journal · doi:10.1111/pbi.12982 · Cited by 235 · see citing works
A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture
O'Geen, H., Henry, I. M., Bhakta, M. S., Meckler, J. F., & Segal, D. J. (2015) · Nucleic Acids Research · doi:10.1093/nar/gkv137 · Cited by 219 · see citing works
Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq
Kim, D., Kim, S., Kim, S., Park, J., & Kim, J.-S. (2016) · Genome Research · doi:10.1101/gr.199588.115 · Cited by 208 · see citing works
Genome-wide target specificity of CRISPR RNA-guided adenine base editors
Kim, D., Kim, D.-E., Lee, G., Cho, S.-I., & Kim, J. (2019) · Nature Biotechnology · doi:10.1038/s41587-019-0050-1 · Cited by 192 · see citing works
DSBCapture: in situ capture and sequencing of DNA breaks
Lensing, S. V., Marsico, G., Hänsel‐Hertsch, R., Lam, E. Y. N., Tannahill, D., & Balasubramanian, S. (2016) · Nature Methods · doi:10.1038/nmeth.3960 · Cited by 155 · see citing works
Fu, Y., Rocha, P. P., Luo, V. M., Raviram, R., Deng, Y., Mazzoni, E. O., & Skok, J. A. (2016) · Nature Communications · doi:10.1038/ncomms11707 · Cited by 148 · see citing works
Genome-wide identification of CRISPR/Cas9 off-targets in human genome
Duan, J., Lu, G., Xie, Z., Lou, M., Luo, J., Guo, L., & Zhang, Y. (2014) · Cell Research · doi:10.1038/cr.2014.87 · Cited by 137 · see citing works
DNA stretching induces Cas9 off-target activity
Newton, M. D., Taylor, B., Driessen, R. P. C., Roos, L., Cvetešić, N., Allyjaun, S., Lenhard, B., Cuomo, M. E., & Rueda, D. (2019) · Nature Structural & Molecular Biology · doi:10.1038/s41594-019-0188-z · Cited by 134 · see citing works
Stabilization of Foxp3 expression by CRISPR-dCas9-based epigenome editing in mouse primary T cells
Okada, M., Kanamori, M., Someya, K., Nakatsukasa, H., & Yoshimura, A. (2017) · Epigenetics & Chromatin · doi:10.1186/s13072-017-0129-1 · Cited by 128 · see citing works
Iannelli, F., Galbiati, A., Capozzo, I., Nguyen, Q. H., Magnuson, B., Michelini, F., D’Alessandro, G., Cabrini, M., Roncador, M., Francia, S., et al. (2017) · Nature Communications · doi:10.1038/ncomms15656 · Cited by 124 · see citing works
Self-citationPrecise and efficient scarless genome editing in stem cells using CORRECT
Kwart, D., Paquet, D., Teo, S., & Tessier‐Lavigne, M. (2017) · Nature Protocols · doi:10.1038/nprot.2016.171 · Cited by 115 · see citing works
DIG-Seq: A genome-wide CRISPR off-target profiling method using chromatin DNA
Kim, D., & Kim, J.-S. (2018) · Genome Research · doi:10.1101/gr.236620.118 · Cited by 108 · see citing works
Defining CRISPR–Cas9 genome-wide nuclease activities with CIRCLE-seq
Lazzarotto, C. R., Nguyen, N. T., Tang, X., Malagon-Lopez, J., Guo, J. A., Aryee, M. J., Joung, J. K., & Tsai, S. Q. (2018) · Nature Protocols · doi:10.1038/s41596-018-0055-0 · Cited by 96 · see citing works
Biased and unbiased methods for the detection of off-target cleavage by CRISPR/Cas9: An overview
Martin, F., Sánchez-Hernández, S., Gutiérrez-Guerrero, A., Pinedo-Gomez, J., & Benabdellah, K. (2016) · International Journal of Molecular Sciences · doi:10.3390/ijms17091507 · Cited by 92 · see citing works
CRISPR–Cas9 a boon or bane: the bumpy road ahead to cancer therapeutics
Ghosh, D., Venkataramani, P., Nandi, S., & Bhattacharjee, S. (2019) · Cancer Cell International · doi:10.1186/s12935-019-0726-0 · Cited by 76 · see citing works
Viral pathogenesis: Unlocking Ebola persistence
Racine, T., & Kobinger, G. (2017) · Nature Microbiology · doi:10.1038/nmicrobiol.2017.124 · Cited by 25 · see citing works
Noncoding transcription influences the replication initiation program through chromatin regulationPreprint
Soudet, J., Gill, J. K., & Stutz, F. (2018) · Genome Research · doi:10.1101/gr.239582.118 · Cited by 23 · see citing works
Identifying a ten-microRNA signature as a superior prognosis biomarker in colon adenocarcinoma
Ma, R., Zhao, Y., He, M., Zhao, H., Zhang, Y., Zhou, S., Gao, M., Di, D., Wang, J., Ding, J., et al. (2019) · Cancer Cell International · doi:10.1186/s12935-019-1074-9 · Cited by 23 · see citing works
Genome-Wide Profiling of DNA Double-Strand Breaks by the BLESS and BLISS Methods
Mirzazadeh, R., Kallas, T., Bienko, M., & Crosetto, N. (2017) · Methods in molecular biology · doi:10.1007/978-1-4939-7306-4_14 · Cited by 18 · see citing works
Self-citationTEG-Seq: An Ion Torrent-Adapted NGS Workflow for in Cellulo Mapping of CRISPR Specificity
Tang, P., Bo, D., Peng, L., Mozhayskiy, V., Potter, J., & Chesnut, J. D. (2018) · BioTechniques · doi:10.2144/btn-2018-0105 · Cited by 11 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Off-target effects in CRISPR/Cas9 gene editing
Guo, C., Ma, X., Gao, F., & Guo, Y. (2023) · Frontiers in Bioengineering and Biotechnology · doi:10.3389/fbioe.2023.1143157 · Cited by 625 · see citing works
“BLISS ligates DSB ends with adapters containing the T7 promoter, so the tagged DNA fragments can be linearly amplified via T7-mediated transcription before sequencing (Yan et al., 2017; Ballarino et al., 2021).”
MethodGenome editing and its applications in genetic improvement in aquaculture
Yang, Z., Yu, Y., Tay, Y. X., & Yue, G. H. (2021) · Reviews in Aquaculture · doi:10.1111/raq.12591 · Cited by 121 · see citing works
Genome-wide detection of DNA double-strand breaks by in-suspension BLISS
Bouwman, B. A. M., Agostini, F., Garnerone, S., Petrosino, G., Gothe, H. J., Sayols, S., Moor, A. E., Itzkovitz, S., Bienko, M., Roukos, V., et al. (2020) · Nature Protocols · doi:10.1038/s41596-020-0397-2 · Cited by 43 · see citing works
Self-citationMoran, M. N., Jones, D. B., Jensen, S. A., Marcoli, R., & Jerry, D. R. (2023) · Reviews in Aquaculture · doi:10.1111/raq.12889 · Cited by 30 · see citing works
Methods for detecting off-target effects of CRISPR/Cas9
Xu, Y., Zhou, S., Wang, L.-Y., Zhang, R., Li, K., Qian, Z., & Li, X. (2025) · Biotechnology Advances · doi:10.1016/j.biotechadv.2025.108750 · Cited by 11 · see citing works
Li, W., Wang, C., & Lu, Y. (2024) · Nano Research · doi:10.1007/s12274-024-6729-8 · Cited by 4 · see citing works
(2020) · Methods in molecular biology · doi:10.1007/978-1-0716-0687-2 · Cited by 3 · see citing works
CRISPR-Based Genome Editing and Delivery Systems: Advances, Challenges and Future Directions
Kaur, A., Chanan, R., Sharma, M., Sharma, S., & Sharma, D. (2026) · IntechOpen eBooks · doi:10.5772/intechopen.1016405
- Authors
- Ballarino, R., Bouwman, B. A. M., & Crosetto, N.
- Affiliations
- Science for Life Laboratory (SE)
- Corresponding author
- Crosetto, Nicola
- Venue
- Methods in molecular biology
- Publisher
- Springer US
- Published
- 2020-09-15
- Volume
- 2162
- Pages
- 261-281
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- OpenAlex
- W3084890671
- Cited by
- 8
- References
- 50
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2023: 2 · 2021: 1 · 2020: 2
- FWCI
- 1.69
- Type
- book chapter
- Language
- en
- Open access
- closed
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- 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 › DNA Repair Mechanisms
- Keywords
- DNA double-strand breaks, genome editing safety, cultured cells
- MeSH terms
- Gene Editing, Humans, Gene Targeting, DNA Breaks, Double-Stranded, HEK293 Cells, CRISPR-Cas Systems
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
