CIRCLE-Seq: A highly sensitive in vitro screen for genome-wide CRISPR-cas9 nuclease off-targets
Open accessAbstract
Sensitive detection of off-target effects is important for translating CRISPR-Cas9 nucleases into human therapeutics. In vitro biochemical methods for finding off-targets offer the potential advantages of greater reproducibility and scalability while avoiding limitations associated with strategies that require the culture and manipulation of living cells. Here we describe circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), a highly sensitive, sequencing-efficient in vitro screening strategy that outperforms existing cell-based or biochemical approaches for identifying CRISPR-Cas9 genome-wide off-target mutations. In contrast to previously described in vitro methods, we show that CIRCLE-seq can be practiced using widely accessible next-generation sequencing technology and does not require reference genome sequences. Importantly, CIRCLE-seq can be used to identify off-target mutations associated with cell-type-specific single-nucleotide polymorphisms, demonstrating the feasibility and importance of generating personalized specificity profiles. CIRCLE-seq provides an accessible, rapid, and comprehensive method for identifying genome-wide off-target mutations of CRISPR-Cas9.
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The false positive rate of CIRCLE-seq is still high and needs careful downstream validation by targeted sequencing.
“However, the false positive rate of CIRCLE-seq is still high and needs careful downstream validation by targeted sequencing (Tsai et al., 2017).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2017
The Discussion recommends using CIRCLE-seq as an initial screen and verifying potential off-target sites with an orthogonal approach; it also describes targeted sequencing used for validation. But it says the true false-positive rate is challenging to estimate, not that it is high. The provided copy gives no page number for these passages. (LLM, conf=0.89)
Experimental — accuracy not yet measuredCIRCLE-seq can detect genome-wide off-target sites without performing WGS.
“CIRCLE-seq is another method that can detect genome-wide off-target sites without performing WGS.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Tsai et al., 2017
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- Authors
- Tsai, S. Q., Nguyen, N. T., Malagon-Lopez, J., Topkar, V. V., Aryee, M. J., & Joung, J. K.
- Affiliations
- Harvard University (US)
- Massachusetts General Hospital (US)
- Corresponding author
- Tsai, Shengdar Q
- Joung, J Keith
- Venue
- Nature Methods
- Published
- 2017-05-01
- Volume
- 14
- Issue
- 6
- Pages
- 607-614
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC5924695
- OpenAlex
- W2611661371
- Cited by
- 814
- References
- 44
- Citations by year
- 2026: 82 · 2025: 84 · 2024: 82 · 2023: 91 · 2022: 86 · 2021: 96 · 2020: 117 · 2019: 77 · 2018: 69 · 2017: 29
- FWCI
- 32.60
- Type
- journal article
- Language
- en
- Update notices
- erratum (2018-05-01)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Nature Methods (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- Funders
- National Institute of General Medical Sciences (DP1GM105378, R35GM118158, R01GM107427)
- 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
- Health Sciences › Medicine › Oncology › CAR-T cell therapy research
- Keywords
- CIRCLE-seq, CRISPR/Cas9, off-target effects, circularization, single nucleotide polymorphisms
- MeSH terms
- Chromosome Mapping, Deoxyribonucleases, Sensitivity and Specificity, Reproducibility of Results, Genome, Sequence Analysis, DNA, High-Throughput Nucleotide Sequencing, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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