Conformational control of DNA target cleavage by CRISPR–Cas9
Open accessAbstract
Cas9 is an RNA-guided DNA endonuclease that targets foreign DNA for destruction as part of a bacterial adaptive immune system mediated by clustered regularly interspaced short palindromic repeats (CRISPR). Together with single-guide RNAs, Cas9 also functions as a powerful genome engineering tool in plants and animals, and efforts are underway to increase the efficiency and specificity of DNA targeting for potential therapeutic applications. Studies of off-target effects have shown that DNA binding is far more promiscuous than DNA cleavage, yet the molecular cues that govern strand scission have not been elucidated. Here we show that the conformational state of the HNH nuclease domain directly controls DNA cleavage activity. Using intramolecular Förster resonance energy transfer experiments to detect relative orientations of the Cas9 catalytic domains when associated with on- and off-target DNA, we find that DNA cleavage efficiencies scale with the extent to which the HNH domain samples an activated conformation. We furthermore uncover a surprising mode of allosteric communication that ensures concerted firing of both Cas9 nuclease domains. Our results highlight a proofreading mechanism beyond initial protospacer adjacent motif (PAM) recognition and RNA–DNA base-pairing that serves as a final specificity checkpoint before DNA double-strand break formation.
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It describes that Cas9 with guide RNA scans for NGG PAMs and, upon sufficient guide-target complementarity, changes conformation and cuts DNA to make a double-strand break.
“Cas9/gRNA is directed to search for NGG protospacer adjacent motifs (PAMs) in the genome and if sufficient complementarity exists between the guide and the target, conformational changes occur and the now fully active Cas9 cleaves the DNA, resulting in a targeted double-stranded break (DSB)5,6.”
From A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells (2018) · cites this paper as [6]
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- Authors
- Sternberg, S. H., LaFrance, B., Kaplan, M., & Doudna, J. A.
- Affiliations
- University of California, Berkeley (US)
- Howard Hughes Medical Institute (US)
- Corresponding author
- Doudna, Jennifer A.
- Venue
- Nature
- Published
- 2015-10-28
- Volume
- 527
- Issue
- 7576
- Pages
- 110-113
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC4859810
- OpenAlex
- W2110058183
- Semantic Scholar
- 2dd6ba89e4bed7e2163200dd205bd41185762089
- Cited by
- 627
- References
- 31
- Citations by year
- 2026: 45 · 2025: 48 · 2024: 58 · 2023: 47 · 2022: 77 · 2021: 71 · 2020: 56 · 2019: 62 · 2018: 54 · 2017: 63 · 2016: 41 · 2015: 2 · 2014: 1 · 2013: 1
- FWCI
- 23.13
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- National Institute of General Medical Sciences (T32GM007232)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- CRISPR/Cas9, DNA cleavage specificity, conformational control, allosteric communication, off-target effects, protospacer adjacent motif, RNA-guided DNA endonuclease, fluorescence resonance energy transfer, genome editing
- MeSH terms
- RNA, Guide, CRISPR-Cas Systems, Allosteric Regulation, Bacterial Proteins, Binding Sites, DNA, Endonucleases, Genetic Engineering, Models, Molecular, Streptococcus pyogenes, Base Pairing, Catalytic Domain, Fluorescence Resonance Energy Transfer, DNA Cleavage, DNA Breaks, Double-Stranded, CRISPR-Cas Systems, CRISPR-Associated Proteins
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