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

Enhanced proofreading governs CRISPR-Cas9 targeting accuracy

Janice S. Chen, Yavuz S. Dagdas, Benjamin P. Kleinstiver, Moira M. Welch, Lucas B. Harrington, Samuel H. Sternberg, J. Keith Joung, Ahmet Yildiz, Jennifer A. Doudna
doi: https://doi.org/10.1101/160036
Janice S. Chen
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yavuz S. Dagdas
2Biophysics Graduate Group, University of California, Berkeley, California 94720, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Benjamin P. Kleinstiver
3Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
4Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Moira M. Welch
3Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lucas B. Harrington
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Samuel H. Sternberg
5Department of Chemistry, University of California, Berkeley, California 94720, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
J. Keith Joung
3Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA
4Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ahmet Yildiz
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA
6Department of Physics, University of California, Berkeley, California 94720, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jennifer A. Doudna
1Department of Molecular and Cell Biology, University of California, Berkeley, California, 94720, USA
5Department of Chemistry, University of California, Berkeley, California 94720, USA
7Howard Hughes Medical Institute, University of California, Berkeley, California 94720, USA
8Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1-4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5-9. Using single-molecule Förster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target mismatches and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we identified residues within REC3 involved in mismatch sensing and designed a new hyper-accurate Cas9 variant (HypaCas9) that retains robust on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.

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 August 12, 2017.
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.
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
(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
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
Janice S. Chen, Yavuz S. Dagdas, Benjamin P. Kleinstiver, Moira M. Welch, Lucas B. Harrington, Samuel H. Sternberg, J. Keith Joung, Ahmet Yildiz, Jennifer A. Doudna
bioRxiv 160036; doi: https://doi.org/10.1101/160036
Reddit logo Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools
Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
Janice S. Chen, Yavuz S. Dagdas, Benjamin P. Kleinstiver, Moira M. Welch, Lucas B. Harrington, Samuel H. Sternberg, J. Keith Joung, Ahmet Yildiz, Jennifer A. Doudna
bioRxiv 160036; doi: https://doi.org/10.1101/160036

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 (4667)
  • Biochemistry (10332)
  • Bioengineering (7652)
  • Bioinformatics (26277)
  • Biophysics (13497)
  • Cancer Biology (10663)
  • Cell Biology (15388)
  • Clinical Trials (138)
  • Developmental Biology (8479)
  • Ecology (12799)
  • Epidemiology (2067)
  • Evolutionary Biology (16817)
  • Genetics (11378)
  • Genomics (15451)
  • Immunology (10591)
  • Microbiology (25139)
  • Molecular Biology (10186)
  • Neuroscience (54316)
  • Paleontology (399)
  • Pathology (1663)
  • Pharmacology and Toxicology (2889)
  • Physiology (4331)
  • Plant Biology (9223)
  • Scientific Communication and Education (1585)
  • Synthetic Biology (2551)
  • Systems Biology (6769)
  • Zoology (1459)