Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs
Open accessAbstract
CRISPR systems have emerged as transformative tools for altering genomes in living cells with unprecedented ease, inspiring keen interest in increasing their specificity for perfectly matched targets. We have developed a novel approach for improving specificity by incorporating chemical modifications in guide RNAs (gRNAs) at specific sites in their DNA recognition sequence ('guide sequence') and systematically evaluating their on-target and off-target activities in biochemical DNA cleavage assays and cell-based assays. Our results show that a chemical modification (2'-O-methyl-3'-phosphonoacetate, or 'MP') incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes. These findings reveal a unique method for enhancing specificity by chemically modifying the guide sequence in gRNAs. Our approach introduces a versatile tool for augmenting the performance of CRISPR systems for research, industrial and therapeutic applications.
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Truncated gRNA truncated by 2-3 bp at the 5' end decreases off-target effects.
“Truncated gRNA: truncated by 2-3bp at 5’ end | Decreased off-target effects”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [73]
The Introduction (page not available) identifies 5′-end gRNA truncation as an approach reported to reduce off-target events. It does not specify truncation by 2–3 bp; the paper’s own results instead concern chemical modifications of sgRNAs. (LLM, conf=0.85)
Experimental — accuracy not yet measuredExtended gRNA GGX20 decreases off-target effects.
“Extended gRNA: GGX20 | Decreased off-target effects”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [73]
The Introduction (page not available) lists 5′ gRNA extensions among approaches reported to increase CRISPR–Cas9 specificity and minimize off-target events. It does not identify the extension as GGX20 or report a result for that specific design; the paper’s Results instead concern chemically modified guides. (LLM, conf=0.83)
Experimental — accuracy not yet measuredMP modifications at certain positions in the ribose-phosphate backbone of sgRNA can enhance on-target specificity while dramatically reducing off-target activity.
“One study incorporated 2’-O-methyl-3’-phosphonoacetate (MP) into the ribose-phosphate backbone of sgRNA and found that MP modifications at certain positions can enhance on-target specificity while dramatically reducing off-target activity”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Ryan et al., 2018
Chemical modifications on sgRNAs can influence their off-target effect.
“Apart from sgRNA length adjustment, chemical modifications on sgRNAs can also influence their off-target effect.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Ryan et al., 2018
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Off-target effects in CRISPR/Cas9 gene editing
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“One study incorporated 2’-O-methyl-3’-phosphonoacetate (MP) into the ribose-phosphate backbone of sgRNA and found that MP modifications at certain positions can enhance on-target specificity while dramatically reducing off-target activity (Ryan et al., 2018).”
BackgroundLatest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing
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- Authors
- Ryan, D. E., Taussig, D., Steinfeld, I., Phadnis, S. M., Lunstad, B. D., Singh, M., Vuong, X., Okochi, K. D., McCaffrey, R., Olesiak, M., Roy, S., Yung, C. W., Curry, B., Sampson, J. R., Bruhn, L., & Dellinger, D. J.
- Affiliations
- Agilent Technologies (United States) (US)
- University of Colorado Boulder (US)
- Corresponding author
- Bruhn, Laurakay
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2017-12-04
- Volume
- 46
- Issue
- 2
- Pages
- 792-803
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- PMC
- PMC5778453
- OpenAlex
- W2772587092
- Cited by
- 280
- References
- 41
- Citations by year
- 2026: 22 · 2025: 29 · 2024: 33 · 2023: 26 · 2022: 46 · 2021: 36 · 2020: 45 · 2019: 30 · 2018: 13
- FWCI
- 9.82
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Nucleic Acids Research (publishedVersion, cc-by-nc)
- PubMed Central (publishedVersion, cc-by-nc)
- 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 Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR-Cas, off-target effects, on-target activity, DNA cleavage specificity, genome editing
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, Base Sequence, Binding Sites, Humans, Phosphonoacetic Acid, K562 Cells, DNA Cleavage, CRISPR-Cas Systems
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