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dc.contributor.author
Pacesa, Martin
dc.contributor.author
Lin, Chun-Han
dc.contributor.author
Cléry, Antoine
dc.contributor.author
Saha, Aakash
dc.contributor.author
Arantes, Pablo R.
dc.contributor.author
Bargsten, Katja
dc.contributor.author
Irby, Matthew J.
dc.contributor.author
Allain, Frédéric H.-T.
dc.contributor.author
Palermo, Giulia
dc.contributor.author
Cameron, Peter
dc.contributor.author
Donohoue, Paul D.
dc.contributor.author
Jinek, Martin
dc.date.accessioned
2022-10-31T09:34:22Z
dc.date.available
2022-10-29T05:39:54Z
dc.date.available
2022-10-31T09:34:22Z
dc.date.issued
2022-10-27
dc.identifier.issn
0092-8674
dc.identifier.issn
1097-4172
dc.identifier.other
10.1016/j.cell.2022.09.026
en_US
dc.identifier.uri
http://hdl.handle.net/20.500.11850/578389
dc.identifier.doi
10.3929/ethz-b-000578389
dc.description.abstract
The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
Cell Press
en_US
dc.rights.uri
http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.subject
CRISPR
en_US
dc.subject
Cas9
en_US
dc.subject
genome editing
en_US
dc.subject
off-target
en_US
dc.subject
nuclease
en_US
dc.subject
guide RNA
en_US
dc.subject
X-ray crystallography
en_US
dc.subject
base pairing
en_US
dc.subject
mismatch
en_US
dc.title
Structural basis for Cas9 off-target activity
en_US
dc.type
Journal Article
dc.rights.license
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
dc.date.published
2022-10-27
ethz.journal.title
Cell
ethz.journal.volume
185
en_US
ethz.journal.issue
22
en_US
ethz.journal.abbreviated
Cell
ethz.pages.start
4067
en_US
ethz.pages.end
4081.e21
en_US
ethz.version.deposit
publishedVersion
en_US
ethz.identifier.wos
ethz.identifier.scopus
ethz.publication.place
Cambridge, MA
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02030 - Dep. Biologie / Dep. of Biology::02517 - Institut für Biochemie / Institute of Biochemistry (IBC)::03591 - Allain, Frédéric / Allain, Frédéric
ethz.leitzahl.certified
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02030 - Dep. Biologie / Dep. of Biology::02517 - Institut für Biochemie / Institute of Biochemistry (IBC)::03591 - Allain, Frédéric / Allain, Frédéric
ethz.date.deposited
2022-10-29T05:39:54Z
ethz.source
SCOPUS
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2022-10-31T09:34:24Z
ethz.rosetta.lastUpdated
2024-02-02T18:50:10Z
ethz.rosetta.versionExported
true
ethz.COinS
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