Extru-seq: A method for predicting genome-wide Cas9 off-target sites with advantages of both cell-based and in vitro approaches
Open accessAbstract
We present a novel genome-wide off-target prediction method named Extru-seq and compare it with cell-based (GUIDE-seq), in vitro (Digenome-seq), and in silico methods using promiscuous guide RNAs with large numbers of valid off-target sites. Extru-seq demonstrates a high validation rate and retention of information about the intracellular environment, both beneficial characteristics of cell-based methods. Extru-seq also shows a low miss rate and could easily be performed in clinically relevant cell types with little optimization, which are major positive features of the in vitro methods. In summary, Extru-seq shows beneficial features of cell-based and in vitro methods.
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Extru-seq has a significantly lower false positive rate than other cell-free methods such as Digenome-seq.
“Compared to other cell-free methods such as Digenome-seq, the false positive rate of Extru-seq is significantly lower (Kwon et al., 2023).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Kwon et al., 2023
The Results section (‘GUIDE-seq and Extru-seq show high validation rates’; page unavailable) reports a significantly higher validation rate for Extru-seq than Digenome-seq among each method’s top 10 predicted sites (92.5% versus 45%). That supports fewer unvalidated predictions in the tested candidates, but the paper does not establish a significantly lower false-positive rate across all predicted sites or all cell-free methods. (LLM, conf=0.84)
Experimental — accuracy not yet measuredExtru-seq exhibited a much lower false negative rate than GUIDE-seq (2.3% versus 29%).
“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 Kwon et al., 2023
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References 42 · Cited by 24
- Authors
- Kwon, J., Kim, M., Hwang, W., Jo, A., Hwang, G.-H., Jung, M., Kim, U. G., Cui, G., Kim, H., Eom, J.-H., Hur, J. K., Lee, J., Kim, Y., Kim, J.-S., Bae, S., & Lee, J. K.
- Affiliations
- ToolGen (South Korea) (KR)
- Hanyang University (KR)
- Yonsei University (KR)
- National Institute of Food and Drug Safety Evaluation (KR)
- Seoul National University (KR)
- Corresponding author
- Lee, Jungjoon K.
- ORCID
- Venue
- Genome biology
- Published
- 2023-01-10
- Volume
- 24
- Issue
- 1
- Pages
- 4-4
- ISSN
- 1465-6906 (ISSN-L)
- 1474-760X
- PMC
- PMC9832775
- OpenAlex
- W4313947391
- Cited by
- 24
- References
- 42
- Citations by year
- 2026: 5 · 2025: 11 · 2024: 7 · 2023: 1
- FWCI
- 2.14
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Genome biology (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Seoul National University Open Repository (Seoul National University) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Ministry of Food and Drug Safety (19172MFDS167)
- 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
- CRISPR/Cas9, GUIDE-seq, intracellular environment
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Genome, CRISPR-Cas Systems
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