BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks
Open accessAbstract
Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.
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For the sgRNA targeting EMX1, BLESS uncovered 6 off-target sites, all of which are included in the 10 genuine off-target sites that BLISS discovered.
“For the sgRNA targeting EMX1, BLESS uncovered 6 off-target sites, all of which are included in the 10 genuine off-target sites that BLISS discovered.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Yan et al., 2017
The Results section, ‘Profiling of Cas9 and Cpf1 specificity’ (page unavailable), says BLISS detected validated off-target sites for the EMX1 and VEGFA guides, including sites previously identified by BLESS, and found additional sites BLESS missed. Our copy does not state that BLESS found exactly six EMX1 off-target sites, that BLISS found exactly ten, or that all six BLESS sites were among them. (LLM, conf=0.86)
Experimental — accuracy not yet measuredFor the sgRNA targeting VEGFA, BLISS identified 27 additional off-target sites that were not found by BLESS.
“Similarly, for the sgRNA targeting VEGFA, besides the 16 off-target sites that were detected by both methods, BLISS identified 27 additional off-target sites that are not found by BLESS (Yan et al., 2017).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Yan et al., 2017
The Results section, “Profiling of Cas9 and Cpf1 specificity” (page unavailable), says BLISS found new off-target sites absent from BLESS when testing guides targeting VEGFA and EMX1. Our copy does not give the claimed count of 27 additional VEGFA sites or confirm the 16 sites shared by both methods. (LLM, conf=0.85)
Experimental — accuracy not yet measuredAAV delivery in vivo may cause accumulation of off-target mutations.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [98]
The Results section (‘Profiling of Cas9 and Cpf1 specificity’; page unavailable) reports validated off-target sites after Cas9 experiments in transfected cells. It does not establish that AAV delivery in vivo causes off-target mutations to accumulate; the cited passage concerns off-target DNA breaks, not that delivery method or time course. (LLM, conf=0.79)
Experimental — accuracy not yet measuredCompared to BLESS, BLISS demands only a few thousand cells and demonstrates higher sensitivity.
“Compared to BLESS, BLISS demands only a few thousand cells and demonstrates a higher sensitivity.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Yan et al., 2017
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 Yan et al., 2017
BLESS demonstrates a false positive rate lower than 1%.
“BLESS demonstrates a false positive rate lower than 1% (Crosetto et al., 2013; Yan et al., 2017; Kim et al., 2019), validating the accuracy of this method.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Yan et al., 2017
The Methods section describes using validated and non-validated Cas9-BLESS off-targets to select a homology-score cutoff, but gives no BLESS false-positive rate. The Results section compares BLESS with BLISS without reporting a rate below 1%. Searches for that rate and for BLESS specificity found no supporting passage in our copy; page numbers were not available. (LLM, conf=0.76)
Experimental — accuracy not yet measuredLNP delivery in vivo eliminates off-target effects.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [98]
The Results section, “Profiling of Cas9 and Cpf1 specificity” (page unavailable), examines off-target breaks detected by BLISS, not whether LNP delivery eliminates them. The Methods section, “Cas or Cpf1 expression constructs and transfections” (page unavailable), describes plasmid transfection with Lipofectamine. Searches for LNP delivery, in vivo degradation, and elimination of off-target effects found no passage addressing the claim. (LLM, conf=0.76)
Experimental — accuracy not yet measuredLNP delivery in vivo can be quickly degraded.
“| Delivery methods improvement | Application in vivo [95–105] | LNP: Can be quickly degraded in vivo | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [98]
The Methods section describes Lipofectamine transfection of cultured cells, while the Results section discusses BLISS detection of CRISPR off-target activity. Searches for LNPs, lipid nanoparticles, and in vivo degradation found no passage addressing whether LNP delivery is quickly degraded in vivo. Page numbers were unavailable in our copy. (LLM, conf=0.76)
Experimental — accuracy not yet measuredAAV delivery in vivo lasts for years in terminally differentiated cell types.
“| Delivery methods improvement | Application in vivo [95–105] | AAV: Last for years in terminally differentiated cell types | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [98]
The Methods (Cells and tissues; page unavailable) and Results (Profiling of endogenous DSBs in primary cells and tissue; page unavailable) describe BLISS experiments in cells and mouse liver, not AAV delivery. Searches for AAV, terminally differentiated cells, and years-long persistence found no passage addressing the claim. (LLM, conf=0.76)
Experimental — accuracy not yet measured
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- Authors
- Yan, W. X., Mirzazadeh, R., Garnerone, S., Scott, D., Schneider, M. W., Kallas, T., Custodio, J., Wernersson, E., Li, Y., Gao, L., Federova, Y., Zetsche, B., Zhang, F., Bienko, M., & Crosetto, N.
- Affiliations
- Broad Institute (US)
- Science for Life Laboratory (SE)
- Corresponding author
- Zhang, Feng
- Bienko, Magda
- Crosetto, Nicola
- ORCID
- Venue
- Nature Communications
- Published
- 2017-05-12
- Volume
- 8
- Issue
- 1
- Pages
- 15058-15058
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC5437291
- OpenAlex
- W2613056759
- Cited by
- 369
- References
- 27
- Citations by year
- 2026: 15 · 2025: 31 · 2024: 35 · 2023: 38 · 2022: 48 · 2021: 52 · 2020: 68 · 2019: 45 · 2018: 20 · 2017: 16
- FWCI
- 16.56
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- Nature
- Funders
- National Science Foundation (T32GM007753)
- Howard Hughes Medical Institute
- New York Stem Cell Foundation
- Svenska Sällskapet för Medicinsk Forskning
- Karolinska Institutet
- Vetenskapsrådet (621-2014-5503, 521-2014-2866)
- Skolkovo Institute of Science and Technology
- Science for Life Laboratory
- National Institutes of Health
- National Institute of Mental Health (DP1MH100706, R01MH110049)
- National Institute of General Medical Sciences (T32GM007753)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomic variations and chromosomal abnormalities
- Keywords
- DNA double-strand breaks, unique molecular identifiers, Cas9, CRISPR/Cas12a, low-input sequencing, cancer cells, embryonic stem cells, liver tissue
- MeSH terms
- Mouse Embryonic Stem Cells, Animals, Cell Line, Gene Expression Regulation, Humans, Liver, Reproducibility of Results, Genomics, Cell Line, Tumor, Mice, DNA Breaks, Double-Stranded, Genome-Wide Association Study, HEK293 Cells, High-Throughput Nucleotide Sequencing, CRISPR-Cas Systems
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