Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

Structural basis for Cas9 off-target activity

View ORCID ProfileMartin Pacesa, Chun-Han Lin, Antoine Cléry, Katja Bargsten, Matthew J. Irby, View ORCID ProfileFrédéric H.T. Allain, Peter Cameron, Paul D. Donohoue, View ORCID ProfileMartin Jinek
doi: https://doi.org/10.1101/2021.11.18.469088
Martin Pacesa
1Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Martin Pacesa
Chun-Han Lin
2Caribou Biosciences, 2929 Seventh Street #105, Berkeley, CA 94710, United States
3LinkedIn, Sunnyvale, CA 94085, United States
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Antoine Cléry
4Institute of Biochemistry, ETH Zurich, Hönggerbergring 64, CH-8093 Zurich, Switzerland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Katja Bargsten
1Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
5leadXpro AG, Villigen, Switzerland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Matthew J. Irby
2Caribou Biosciences, 2929 Seventh Street #105, Berkeley, CA 94710, United States
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Frédéric H.T. Allain
4Institute of Biochemistry, ETH Zurich, Hönggerbergring 64, CH-8093 Zurich, Switzerland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Frédéric H.T. Allain
Peter Cameron
2Caribou Biosciences, 2929 Seventh Street #105, Berkeley, CA 94710, United States
6Spotlight Therapeutics, Hayward, CA 94545, United States
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Paul D. Donohoue
2Caribou Biosciences, 2929 Seventh Street #105, Berkeley, CA 94710, United States
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Martin Jinek
1Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Martin Jinek
  • For correspondence: jinek@bioc.uzh.ch
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

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 non-canonical base pairing interactions and preservation of base stacking within the guide–off-target heteroduplex. Off-target sites containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping rather than RNA bulge formation. Additionally, PAM-distal mismatches result in duplex unpairing and induce a conformational change of 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.

Competing Interest Statement

P.D.D. and M.J.I are current employees of Caribou Biosciences, Inc., and C-H.L. and P.C. are former employees of Caribou Biosciences, Inc. M.J. is a co-founder of Caribou Biosciences, Inc. M.J., C-H.L., M.J.I., P.C. and P.D.D. are named inventors on patents and patent applications related to CRISPR-Cas technologies.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted November 18, 2021.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Structural basis for Cas9 off-target activity
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Structural basis for Cas9 off-target activity
Martin Pacesa, Chun-Han Lin, Antoine Cléry, Katja Bargsten, Matthew J. Irby, Frédéric H.T. Allain, Peter Cameron, Paul D. Donohoue, Martin Jinek
bioRxiv 2021.11.18.469088; doi: https://doi.org/10.1101/2021.11.18.469088
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
Structural basis for Cas9 off-target activity
Martin Pacesa, Chun-Han Lin, Antoine Cléry, Katja Bargsten, Matthew J. Irby, Frédéric H.T. Allain, Peter Cameron, Paul D. Donohoue, Martin Jinek
bioRxiv 2021.11.18.469088; doi: https://doi.org/10.1101/2021.11.18.469088

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Biochemistry
Subject Areas
All Articles
  • Animal Behavior and Cognition (4676)
  • Biochemistry (10347)
  • Bioengineering (7660)
  • Bioinformatics (26310)
  • Biophysics (13508)
  • Cancer Biology (10672)
  • Cell Biology (15424)
  • Clinical Trials (138)
  • Developmental Biology (8490)
  • Ecology (12808)
  • Epidemiology (2067)
  • Evolutionary Biology (16837)
  • Genetics (11383)
  • Genomics (15471)
  • Immunology (10604)
  • Microbiology (25188)
  • Molecular Biology (10211)
  • Neuroscience (54401)
  • Paleontology (401)
  • Pathology (1667)
  • Pharmacology and Toxicology (2890)
  • Physiology (4335)
  • Plant Biology (9237)
  • Scientific Communication and Education (1586)
  • Synthetic Biology (2556)
  • Systems Biology (6774)
  • Zoology (1461)