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High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells
Open accessAbstract
Clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nucleases (RGNs) have rapidly emerged as a facile and efficient platform for genome editing. Here, we use a human cell-based reporter assay to characterize off-target cleavage of CRISPR-associated (Cas)9-based RGNs. We find that single and double mismatches are tolerated to varying degrees depending on their position along the guide RNA (gRNA)-DNA interface. We also readily detected off-target alterations induced by four out of six RGNs targeted to endogenous loci in human cells by examination of partially mismatched sites. The off-target sites we identified harbored up to five mismatches and many were mutagenized with frequencies comparable to (or higher than) those observed at the intended on-target site. Our work demonstrates that RGNs can be highly active even with imperfectly matched RNA-DNA interfaces in human cells, a finding that might confound their use in research and therapeutic applications.
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In the citing paper
“Moreover, in-depth studies on the outcomes of Cas9-induced double-strand break (DSB) repair have shown significant off-target effects, which resulted in substitution, insertion, deletion, and DNA translocation.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [13]
Experimental — accuracy not yet measuredThe cited source says what the claim attributes to it.
In the citing paper
“Defective off-target binding and cleaving can result in collateral mutagenesis induced by the error-prone repair of DSB by NHEJ [106,107].”
From CRISPR-Cas: Converting A Bacterial Defence Mechanism into A State-of-the-Art Genetic Manipulation Tool (2019) · cites this paper as [107]
The check wasn’t confident either way, so it gives no verdict — never disputed.
In the citing paper
“For example, a study demonstrated that manipulating Cas9's biophysical attributes, such as minimizing nonspecific RNA-DNA interactions, significantly reduces off-target mutations while maintaining high on-target efficiency.”
From Hybrid Neural Networks for Off-Target Prediction in Gene Editing: Integrating Sequence and Biophysical Features (2025) · cites this paper as [5]
Experimental — accuracy not yet measuredIn the citing paper
“A study reported highfrequency off-target mutagenesis in transduced human cell lines after SpCas9 delivery [108].”
From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [108]
In the citing paper
“Using a VEGFA sgRNA with well-characterized off-target sites24, we observed that transient Cas9 expression by mRNA delivery can substantially reduce off-target genome editing in cells (Fig. 1f–h).”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [24]
In the citing paper
“as mentioned in several recent publications, Cas9 tolerates mismatches between the guide RNA and target DNA in a sequence-dependent manner and potentially tolerates up to five target mismatches in human cells [12], [24], [25], [26], [27], thereby promoting undesired off-target mutations.”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [24]
In the citing paper
“non-specific cleavage may occur in the cellular genome [12], [24], [25], [26], [27].”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [24]
In the citing paper
“Because the sequence specificity of Streptococcus pyogenes Cas9 tolerates 4 mismatches between the gRNA and complementary sequence”
From High-efficiency targeted editing of large viral genomes by RNA-guided nucleases (2014) · cites this paper as [24]
In the citing paper
“Collectively, these studies indicate that although each base within the 20 nt guide sequence contributes to overall specificity”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [90]
In the citing paper
“However, it has been recently shown that CRISPR/Cas9 system can be highly active even when mismatches occur between gRNA and their DNA targets”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [90]
In the citing paper
“multiple mismatches between the guide RNA and its complementary target DNA sequence can be tolerated depending on the quantity, position, and base identity of mismatches [87,90,91,93], all of which contribute to potential off-target activity.”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [90]
In the citing paper
“and off-target mutations occur at endogenous loci in human cells with high frequency [90,91,92].”
From Newer Gene Editing Technologies toward HIV Gene Therapy (2013) · cites this paper as [90]
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Citations
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Metadata
- Authors
- Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D.
- Affiliations
- Harvard University (US)
- Massachusetts General Hospital (US)
- Corresponding author
- Joung, J Keith
- Sander, Jeffry D
- Venue
- Nature Biotechnology
- Published
- 2013-06-23
- Volume
- 31
- Issue
- 9
- Pages
- 822-826
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3773023
- OpenAlex
- W2077659966
- Cited by
- 3,292
- References
- 20
- Citations by year
- 2026: 140 · 2025: 181 · 2024: 206 · 2023: 208 · 2022: 246 · 2021: 254 · 2020: 280 · 2019: 274 · 2018: 278 · 2017: 269 · 2016: 314 · 2015: 254 · 2014: 291 · 2013: 90
- FWCI
- 141.80
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Nature Biotechnology (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 Human Genome Research Institute (P50HG005550)
- National Institute of General Medical Sciences (DP1GM105378, R01GM088040)
- National Center for Chronic Disease Prevention and Health Promotion (DP1 GM105378)
- 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, off-target cleavage, RNA-guided nucleases, mismatch tolerance, genome editing, mutagenesis, human cells, off-target sites
- MeSH terms
- Base Sequence, Endonucleases, Genetic Engineering, Humans, Molecular Sequence Data, Mutagenesis, K562 Cells, HEK293 Cells, CRISPR-Associated Proteins
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