Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors
Abstract
Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.
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Detect-seq chemically labels deoxyuridine, the deamination product of cytosine, and enriches CBE edited genomic loci by biotin-streptavidin pulldown for deep sequencing analysis.
“More specifically, Detect-seq chemically labels deoxyuridine, the deamination product of cytosine, and enriches CBE edited genomic loci by biotin-streptavidin pulldown for deep sequencing analysis”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Lei et al., 2021
EndoV-seq and Detect-seq have greatly facilitated detection of off-target effects of ABE and CBE on genomic DNA.
“The recent development of EndoV-seq and Detect-seq have greatly facilitated the detection of off-target effects of ABE and CBE on genomic DNA.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Lei et al., 2021
Detect-seq measures CBE off-target effects by tracking its reaction intermediates.
“Similar to EndoV-seq, Detect-seq measures CBE off-target effects by tracking its reaction intermediates”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Lei et al., 2021
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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
“Similar to EndoV-seq, Detect-seq measures CBE off-target effects by tracking its reaction intermediates (Lei et al., 2021).”
BackgroundMethodPrecise plant genome editing using base editors and prime editors
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- Authors
- Lei, Z., Meng, H., Lv, Z., Liu, M., Zhao, H., Wu, H., Zhang, X., Liu, L., Zhuang, Y., Yin, K., Yan, Y., & Yi, C.
- Affiliations
- Peking University (CN)
- Corresponding author
- Yi, Chengqi
- ORCID
- Venue
- Nature Methods
- Published
- 2021-06
- Volume
- 18
- Issue
- 6
- Pages
- 643-651
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- OpenAlex
- W3172701693
- Cited by
- 67
- References
- 47
- Citations by year
- 2026: 10 · 2025: 15 · 2024: 11 · 2023: 14 · 2022: 12 · 2021: 5
- FWCI
- 3.29
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Nature Methods (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 and Nucleic Acid Chemistry
- Keywords
- cytosine base editors, off-target editing, chemical labeling, single guide RNA, genome editing tools
- MeSH terms
- Gene Editing, Whole Genome Sequencing, Cytosine, Humans, Mutation, RNA, MCF-7 Cells, CRISPR-Cas Systems
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