DNA targeting specificity of RNA-guided Cas9 nucleases
Open accessAbstract
The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
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Off-target cleavages of Cas9 nucleases that cause oncogenic mutations in the genome have been reported extensively.
“For example, off-target cleavages of the Cas9 nucleases that cause oncogenic mutations in the genome have been reported extensively [123,124].”
From Utility of iPSC-Derived Cells for Disease Modeling, Drug Development, and Cell Therapy (2022) · cites this paper as [123]
The paper’s Abstract reports SpCas9-induced indel mutations at predicted genomic off-target loci in human cells, supporting the off-target cleavage portion of the claim. It does not identify those mutations as oncogenic or report that oncogenic mutations from Cas9 off-target cleavage have been extensively documented. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThe off-target sites are often sgRNA-dependent, since Cas9 is known to tolerate up to 3 mismatches between sgRNA and genomic DNA.
“The off-target sites are often sgRNA-dependent, since Cas9 is known to tolerate up to 3 mismatches between sgRNA and genomic DNA (Fu et al., 2013; Hsu et al., 2013; Wang et al., 2016a).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Hsu et al., 2013
The passages indexed under Abstract (no page provided) report sgRNA-dependent off-target cleavage and show that mismatch number and position affect Cas9 activity. They do not support an unqualified three-mismatch tolerance: three or more interspaced mismatches eliminated detectable cleavage at the vast majority of loci studied. (LLM, conf=0.84)
Experimental — accuracy not yet measuredOff-target effects remain a major challenge for CRISPR/Cas systems in translational medicine.
“Although CRISPR/Cas systems exhibit tremendous potential in translational medicine, off-target effects remain a major challenge (Fu et al., 2013; Hsu et al., 2013; Pacesa et al., 2022).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Hsu et al., 2013
The paper’s Abstract (page unavailable) documents off-target editing in human cells and describes ways to minimize it, supporting the claim that off-target effects are a challenge for Cas9 genome editing. It does not specifically assess translational medicine or establish how major that challenge is in clinical use. (LLM, conf=0.81)
Experimental — accuracy not yet measuredThe MIT algorithm weights the position effect of mismatches in the sgRNA to generate a score to evaluate off-target effects.
“The algorithm of MIT weights the position effect of the mismatches in the sgRNA to generate a score to evaluate off-target effects (Hsu et al., 2013; Haeussler et al., 2016).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Hsu et al., 2013
The PAM sequence adjacent to the target sequence greatly affects the editing efficiency and editing range of Cas9.
“The PAM sequence adjacent to the target sequence greatly affects the editing efficiency and editing range of Cas9.”
From Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing (2024) · cites this paper as [2]
EvoCas9-1 paired with Evo-generated sgRNA had in vitro cleavage activity comparable to SpCas9 paired with canonical SpCas9 sgRNA.
“Recombinant expression and purification of EvoCas9–1 paired with chemically synthesized Evo-generated single guide RNA (sgRNA) exhibited comparable in vitro cleavage activity to SpCas9 paired with the canonical SpCas9 sgRNA”
From Sequence modeling and design from molecular to genome scale with Evo (2024) · cites this paper as [63]
The paper discusses SpCas9 guide-RNA architecture and cleavage activity, and its Online Methods describe an in vitro SpCas9 cleavage assay (page unavailable). Our copy does not address EvoCas9-1 or an Evo-generated sgRNA, so it cannot verify the claimed comparison; searches for those terms and for chemically synthesized sgRNA found no matching passage. (LLM, conf=0.78)
Experimental — accuracy not yet measuredPaired SpCas9 nickases can create DSBs with markedly lower off-target effects when two sgRNAs are designed to simultaneously cut the two opposite strands of DNA within a small distance.
“When two sgRNAs are designed to simultaneously cut the two opposite strands of DNA within a small distance, DSBs can be created with markedly lower off-target effects (Hsu et al., 2013; Frock et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Hsu et al., 2013
The passage on the D10A nickase (section labeled Abstract; page unavailable) describes editing with a single guide and no detectable on-target indels. Searches for paired nickases and guides targeting opposite strands found no passage in our copy addressing whether that arrangement creates double-strand breaks with lower off-target effects. (LLM, conf=0.76)
Experimental — accuracy not yet measuredBy bioinformatic screening and efficiency/off-target prediction, four gRNA targets (LTRs A-D) within the HIV-1 LTR promoter U3 region were identified that avoid conserved transcription factor binding sites, minimizing the likelihood of altering host gene expression.
“By bioinformatic screening and efficiency/off-target prediction (14, 15), we identified four gRNA targets (protospacers; LTRs A-D) that avoid conserved transcription factor binding sites, minimizing the likelihood of altering host gene expression”
From RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection (2014) · cites this paper as [14]
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, thereby promoting undesired off-target mutations.
“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 [25]
The sequence specificity of Streptococcus pyogenes Cas9 tolerates 4 mismatches between the gRNA and complementary sequence.
“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 [25]
Non-specific cleavage may occur in the cellular genome.
“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 [25]
CRISPR/Cas9 system can be highly active even when mismatches occur between gRNA and their DNA targets.
“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 [91]
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, contributing to potential off-target activity.
“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 [91]
Off-target mutations occur at endogenous loci in human cells with high frequency.
“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 [91]
Each base within the 20 nt guide sequence contributes to overall specificity.
“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 [91]
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Self-citation
- Authors
- Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., Cradick, T. J., Marraffini, L. A., Bao, G., & Zhang, F.
- Affiliations
- Broad Institute (US)
- Georgia Institute of Technology (US)
- Koch Institute for Integrative Cancer Research At MIT (US)
- Rockefeller University (US)
- Corresponding author
- Zhang, Feng
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2013-07-21
- Volume
- 31
- Issue
- 9
- Pages
- 827-832
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3969858
- OpenAlex
- W2036176224
- Cited by
- 4,741
- References
- 29
- Citations by year
- 2026: 242 · 2025: 300 · 2024: 304 · 2023: 331 · 2022: 372 · 2021: 424 · 2020: 416 · 2019: 422 · 2018: 463 · 2017: 438 · 2016: 402 · 2015: 297 · 2014: 269 · 2013: 53
- FWCI
- 152.56
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion)
- PMC
- Funders
- National Science Foundation
- Howard Hughes Medical Institute
- Damon Runyon Cancer Research Foundation
- McKnight Foundation
- Life Sciences Research Foundation
- National Institutes of Health (R01‐grant, GM34277)
- Harvard Stem Cell Institute
- National Institute of Mental Health (DP1MH100706)
- National Eye Institute (PN2EY018244)
- National Cancer Institute (R01CA133404)
- National Institute of General Medical Sciences (R01GM034277, R01 GM34277, T32GM007753)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK097768)
- National Center for Chronic Disease Prevention and Health Promotion (DP1‑MH100706)
- 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
- SpCas9, single guide RNA, genome editing, off-target effects, mismatch tolerance, DNA methylation, indel mutations, guide RNA design, target site selection, HEK293T cells, web-based design tool
- MeSH terms
- Bacterial Proteins, Base Sequence, Deoxyribonucleases, DNA, Genetic Engineering, Molecular Sequence Data, Streptococcus pyogenes, Transcription Factors, Base Pair Mismatch, RNA, Small Untranslated
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