Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage
Open accessAbstract
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to (5')NGG(3') protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5' end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.
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Other publications observed a much lower validation rate for dCas9 ChIP-Seq.
“However, other publications observed a much lower validation rate (Cencic et al., 2014; Duan et al., 2014; Tsai et al., 2015).”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cencic et al., 2014
Cas9 could associate with genomic loci without exerting its nuclease function, which is a major caveat in Cas9 ChIP-Seq-based off-target detection assays.
“These studies indicated that Cas9 could associate with genomic loci without exerting its nuclease function, deposing a major caveat in Cas9 ChIP-Seq-based off-target detection assay.”
From Off-target effects in CRISPR/Cas9 gene editing (2023) · cites this paper as Cencic et al., 2014
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- Authors
- Cencic, R., Miura, H., Malina, A., Robert, F., Ethier, S., Schmeing, T. M., Dostie, J., & Pelletier, J.
- Affiliations
- McGill University (CA)
- Institut du cancer Rosalind & Morris Goodman (CA)
- Corresponding author
- Pelletier, Jerry
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2014-10-02
- Volume
- 9
- Issue
- 10
- Pages
- e109213
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC4183563
- OpenAlex
- W1970617888
- Cited by
- 117
- References
- 33
- Citations by year
- 2026: 7 · 2025: 5 · 2024: 10 · 2023: 4 · 2022: 10 · 2021: 9 · 2020: 5 · 2019: 5 · 2018: 6 · 2017: 16 · 2016: 18 · 2015: 18 · 2014: 3 · 2013: 1
- FWCI
- 7.51
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- PLoS ONE (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- eScholarship@McGill (McGill) (submittedVersion, cc-by)
- Figshare (submittedVersion, cc-by)
- Funders
- Canadian Institutes of Health Research (MOP106615, 106530_1, 86716_1, 106615)
- 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 › Chromatin Remodeling and Cancer
- Keywords
- CRISPR/Cas9, protospacer adjacent motif, off-target editing, target specificity, single guide RNA, Trp53, seed region, genome-wide binding, ChIP-seq, genome editing
- MeSH terms
- Animals, Base Sequence, Cells, Cultured, Molecular Sequence Data, Sequence Homology, Nucleic Acid, Chromatin Immunoprecipitation, Mice, DNA Cleavage, Clustered Regularly Interspaced Short Palindromic Repeats
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