Cas9-chromatin binding information enables more accurate CRISPR off-target prediction
Open accessAbstract
The CRISPR system has become a powerful biological tool with a wide range of applications. However, improving targeting specificity and accurately predicting potential off-targets remains a significant goal. Here, we introduce a web-based CR: ISPR/Cas9 O: ff-target P: rediction and I: dentification T: ool (CROP-IT) that performs improved off-target binding and cleavage site predictions. Unlike existing prediction programs that solely use DNA sequence information; CROP-IT integrates whole genome level biological information from existing Cas9 binding and cleavage data sets. Utilizing whole-genome chromatin state information from 125 human cell types further enhances its computational prediction power. Comparative analyses on experimentally validated datasets show that CROP-IT outperforms existing computational algorithms in predicting both Cas9 binding as well as cleavage sites. With a user-friendly web-interface, CROP-IT outputs scored and ranked list of potential off-targets that enables improved guide RNA design and more accurate prediction of Cas9 binding or cleavage sites.
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CCTop and CROP-IT generate scores based on the distances of the mismatches to the PAM.
“CCTop and CROP-IT generate scores based on the distances of the mismatches to the PAM (Singh et al., 2015; Stemmer et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Singh et al., 2015
Chemical modification of gRNA by replacing RNA nucleotides with DNA nucleotides increases on-target efficiency and decreases off-target effects.
“| gRNA improvement | Chemical modifications [76–78] | Replacement of RNA nucleotides into DNA nucleotides | Increased on-target efficiency and decreased off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [76]
The Introduction and Results (page unavailable) discuss computational prediction of Cas9 off-target binding and cleavage, not chemical modification of guide RNA. Searches for RNA-to-DNA nucleotide replacement and its effects on on-target efficiency and off-target activity found no passage addressing the claim. (LLM, conf=0.76)
Experimental — accuracy not yet measuredIncorporation of MP/bridged nucleic acids/locked nucleic acids increases on-target efficiency and decreases off-target effects.
“Incorporation of MP/bridged nucleic acids/locked nucleic acids | Increased on-target efficiency and decreased off-target effects”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [76]
The Results and Methods passages describe CROP-IT, a computational tool for predicting Cas9 off-target sites, not the incorporation of modified nucleic acids. A search of our copy for MP, bridged nucleic acids, locked nucleic acids, and guide-RNA chemical modifications found no passage assessing their effects on on-target efficiency or off-target activity. Page numbers were not available in the indexed passages. (LLM, conf=0.75)
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- Authors
- Singh, R., Kuscu, C., Quinlan, A., Qi, Y., & Adli, M.
- Affiliations
- University of Virginia (US)
- Corresponding author
- Adli, Mazhar
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2015-06-01
- Volume
- 43
- Issue
- 18
- Pages
- e118-e118
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC4605288
- OpenAlex
- W2139314409
- Cited by
- 217
- References
- 39
- Citations by year
- 2026: 7 · 2025: 12 · 2024: 21 · 2023: 14 · 2022: 20 · 2021: 21 · 2020: 29 · 2019: 24 · 2018: 23 · 2017: 20 · 2016: 19 · 2015: 7
- FWCI
- 9.98
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nucleic Acids Research (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health
- National Institute of Diabetes and Digestive and Kidney Diseases (P50DK096373)
- 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 › Genomics and Chromatin Dynamics
- Keywords
- CRISPR/Cas9, off-target prediction, cleavage site prediction, chromatin state, guide RNA design, targeting specificity, web-based prediction tool, human cell types
- MeSH terms
- Algorithms, Binding Sites, Chromatin, Deoxyribonucleases, Humans, Software, Sequence Analysis, DNA, Sequence Analysis, RNA, DNA Cleavage, CRISPR-Cas Systems, CRISPR-Associated Proteins
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