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Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
Open accessAbstract
Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.
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“| Delivery methods improvement | Application in cell culture [89–94] | Viral transduction | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [93]
Experimental — accuracy not yet measuredThe source backs part of the claim, not all of it — or backs it for a different population, condition or comparison.
In the citing paper
“| Delivery methods improvement | Application in cell culture [89–94] | Plasmid transfection | May cause accumulation of off-target mutations |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [93]
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“| Delivery methods improvement | Application in cell culture [89–94] | RNP electroporation: Rapid transfection and turnover | Eliminate off-target effects |”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as [93]
Experimental — accuracy not yet measuredIn the citing paper
“A key feature of our system is the transient expression of the nuclease Cas9, through delivery of mRNA. This allowed for sufficient exposure to the nuclease for gene repair and reduced concerns associated with long-term nuclease exposure34.”
From Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo (2016) · cites this paper as [34]
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“And moreover, DNA binding and cleavage by Cas9 are in some cases uncoupled, that is, Cas9 can bind to but not cleave DNA sequences that are partially complementary to sgRNA 49 and exert epigenomic regulatory effects, 43 44 which is unpredictable by silicon methods relying on base pairing.”
From CRISPR-Cas9: a new and promising player in gene therapy (2015) · cites this paper as [49]
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Metadata
- Authors
- Wu, X., Scott, D. A., Kriz, A. J., Chiu, A. C., Hsu, P. D., Dadon, D. B., Cheng, A. W., Trevino, A. E., Konermann, S., Chen, S., Jaenisch, R., Zhang, F., & Sharp, P. A.
- Affiliations
- Koch Institute for Integrative Cancer Research At MIT (US)
- Broad Institute (US)
- Massachusetts Institute of Technology (US)
- Whitehead Institute for Biomedical Research (US)
- Corresponding author
- Zhang, Feng
- Sharp, Phillip A
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2014-04-20
- Volume
- 32
- Issue
- 7
- Pages
- 670-676
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC4145672
- OpenAlex
- W1506494596
- Cited by
- 944
- References
- 47
- Citations by year
- 2026: 26 · 2025: 42 · 2024: 56 · 2023: 61 · 2022: 73 · 2021: 63 · 2020: 75 · 2019: 80 · 2018: 81 · 2017: 97 · 2016: 123 · 2015: 111 · 2014: 54
- FWCI
- 52.65
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- eScholarship (California Digital Library) (submittedVersion)
- PMC
- Funders
- Howard Hughes Medical Institute
- National Institute of Mental Health (DP1MH100706)
- National Cancer Institute (R01CA133404, P01CA042063, P30CA014051, P30-CA-14051, P01 CA 42063)
- National Institute of General Medical Sciences (R01GM034277, R01 GM34277, R37GM034277, T32GM007287)
- National Institute of Diabetes and Digestive and Kidney Diseases (R01DK097768)
- National Center for Chronic Disease Prevention and Health Promotion (1DP1-MH100706)
- Eunice Kennedy Shriver National Institute of Child Health and Human Development (R37HD045022)
- 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
- Cas9, CRISPR/Cas9, dCas9, genome-wide binding sites, single guide RNA, Streptococcus pyogenes, mouse embryonic stem cells, off-target binding, seed sequence, protospacer adjacent motif, chromatin accessibility, DNA cleavage, target specificity
- MeSH terms
- Animals, Base Sequence, Binding Sites, Cells, Cultured, Deoxyribonuclease I, DNA-Binding Proteins, Models, Genetic, Molecular Sequence Data, Protein Binding, Genome, Mice, Embryonic Stem Cells, CRISPR-Cas Systems
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