skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling

Abstract

The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage.more » Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.« less

Authors:
 [1];  [2];  [2];  [1];  [3];  [1]
  1. Stanford University, Stanford, CA (United States)
  2. Univ. of California, Berkeley, CA (United States)
  3. Univ. of California, Berkeley, CA (United States). Howard Hughes Medical Institute
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH); Defense Advanced Research Projects Agency (DARPA); National Science Foundation (NSF)
OSTI Identifier:
1633265
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 117; Journal Issue: 11; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; magnetic tweezers; gene editing; torque spectroscopy

Citation Formats

Ivanov, Ivan E., Wright, Addison V., Cofsky, Joshua C., Aris, Kevin D. Palacio, Doudna, Jennifer A., and Bryant, Zev. Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling. United States: N. p., 2020. Web. https://doi.org/10.1073/pnas.1913445117.
Ivanov, Ivan E., Wright, Addison V., Cofsky, Joshua C., Aris, Kevin D. Palacio, Doudna, Jennifer A., & Bryant, Zev. Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling. United States. https://doi.org/10.1073/pnas.1913445117
Ivanov, Ivan E., Wright, Addison V., Cofsky, Joshua C., Aris, Kevin D. Palacio, Doudna, Jennifer A., and Bryant, Zev. Mon . "Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling". United States. https://doi.org/10.1073/pnas.1913445117. https://www.osti.gov/servlets/purl/1633265.
@article{osti_1633265,
title = {Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling},
author = {Ivanov, Ivan E. and Wright, Addison V. and Cofsky, Joshua C. and Aris, Kevin D. Palacio and Doudna, Jennifer A. and Bryant, Zev},
abstractNote = {The CRISPR-Cas9 nuclease has been widely repurposed as a molecular and cell biology tool for its ability to programmably target and cleave DNA. Cas9 recognizes its target site by unwinding the DNA double helix and hybridizing a 20-nucleotide section of its associated guide RNA to one DNA strand, forming an R-loop structure. A dynamic and mechanical description of R-loop formation is needed to understand the biophysics of target searching and develop rational approaches for mitigating off-target activity while accounting for the influence of torsional strain in the genome. Here we investigate the dynamics of Cas9 R-loop formation and collapse using rotor bead tracking (RBT), a single-molecule technique that can simultaneously monitor DNA unwinding with base-pair resolution and binding of fluorescently labeled macromolecules in real time. By measuring changes in torque upon unwinding of the double helix, we find that R-loop formation and collapse proceed via a transient discrete intermediate, consistent with DNA:RNA hybridization within an initial seed region. Using systematic measurements of target and off-target sequences under controlled mechanical perturbations, we characterize position-dependent effects of sequence mismatches and show how DNA supercoiling modulates the energy landscape of R-loop formation and dictates access to states competent for stable binding and cleavage. Consistent with this energy landscape model, in bulk experiments we observe promiscuous cleavage under physiological negative supercoiling. The detailed description of DNA interrogation presented here suggests strategies for improving the specificity and kinetics of Cas9 as a genome engineering tool and may inspire expanded applications that exploit sensitivity to DNA supercoiling.},
doi = {10.1073/pnas.1913445117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 11,
volume = 117,
place = {United States},
year = {2020},
month = {3}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 2 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage
journal, January 2016


The new frontier of genome engineering with CRISPR-Cas9
journal, November 2014


Genome Editing B.C. (Before CRISPR): Lasting Lessons from the “Old Testament”
journal, February 2018


Conformational control of DNA target cleavage by CRISPR–Cas9
journal, October 2015

  • Sternberg, Samuel H.; LaFrance, Benjamin; Kaplan, Matias
  • Nature, Vol. 527, Issue 7576
  • DOI: 10.1038/nature15544

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9
journal, January 2014

  • Sternberg, Samuel H.; Redding, Sy; Jinek, Martin
  • Nature, Vol. 507, Issue 7490
  • DOI: 10.1038/nature13011

High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding
journal, May 2017

  • Boyle, Evan A.; Andreasson, Johan O. L.; Chircus, Lauren M.
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 21
  • DOI: 10.1073/pnas.1700557114

Investigating DNA supercoiling in eukaryotic genomes
journal, April 2017

  • Corless, Samuel; Gilbert, Nick
  • Briefings in Functional Genomics, Vol. 16, Issue 6
  • DOI: 10.1093/bfgp/elx007

CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea
journal, June 2013


CRISPR/Cas9 in Genome Editing and Beyond
journal, June 2016


Kinetics of dCas9 target search in Escherichia coli
journal, September 2017


Structural transitions and elasticity from torque measurements on DNA
journal, July 2003

  • Bryant, Zev; Stone, Michael D.; Gore, Jeff
  • Nature, Vol. 424, Issue 6946
  • DOI: 10.1038/nature01810

A transformation for the mechanical fingerprints of complex biomolecular interactions
journal, September 2013

  • Zhang, Y.; Dudko, O. K.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 41
  • DOI: 10.1073/pnas.1309101110

Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection
journal, March 2015


Transcription Under Torsion
journal, June 2013


Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease
journal, November 2016

  • Lim, Youngbin; Bak, So Young; Sung, Keewon
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms13350

The effect of force on thermodynamics and kinetics of single molecule reactions
journal, December 2002


A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture
journal, February 2015

  • O'Geen, Henriette; Henry, Isabelle M.; Bhakta, Mital S.
  • Nucleic Acids Research, Vol. 43, Issue 6
  • DOI: 10.1093/nar/gkv137

Development and Applications of CRISPR-Cas9 for Genome Engineering
journal, June 2014


Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase
journal, January 2001

  • Harada, Yoshie; Ohara, Osamu; Takatsuki, Akira
  • Nature, Vol. 409, Issue 6816
  • DOI: 10.1038/35051126

Ten years of tension: single-molecule DNA mechanics
journal, January 2003

  • Bustamante, Carlos; Bryant, Zev; Smith, Steven B.
  • Nature, Vol. 421, Issue 6921
  • DOI: 10.1038/nature01405

Cas9 as a versatile tool for engineering biology
journal, September 2013

  • Mali, Prashant; Esvelt, Kevin M.; Church, George M.
  • Nature Methods, Vol. 10, Issue 10
  • DOI: 10.1038/nmeth.2649

Enhanced proofreading governs CRISPR–Cas9 targeting accuracy
journal, September 2017

  • Chen, Janice S.; Dagdas, Yavuz S.; Kleinstiver, Benjamin P.
  • Nature, Vol. 550, Issue 7676
  • DOI: 10.1038/nature24268

Off-target Effects in CRISPR/Cas9-mediated Genome Engineering
journal, January 2015

  • Zhang, Xiao-Hui; Tee, Louis Y.; Wang, Xiao-Gang
  • Molecular Therapy - Nucleic Acids, Vol. 4
  • DOI: 10.1038/mtna.2015.37

Genomes in Focus: Development and Applications of CRISPR-Cas9 Imaging Technologies
journal, February 2018

  • Knight, Spencer C.; Tjian, Robert; Doudna, Jennifer A.
  • Angewandte Chemie International Edition, Vol. 57, Issue 16
  • DOI: 10.1002/anie.201709201

The Development of an Information Criterion for Change-Point Analysis
journal, March 2016


Multimodal Measurements of Single-Molecule Dynamics Using FluoRBT
journal, January 2018


A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012


Cellular roles of DNA topoisomerases: a molecular perspective
journal, June 2002

  • Wang, James C.
  • Nature Reviews Molecular Cell Biology, Vol. 3, Issue 6
  • DOI: 10.1038/nrm831

A CRISPR–dCas Toolbox for Genetic Engineering and Synthetic Biology
journal, January 2019


Recent developments in single-molecule DNA mechanics
journal, June 2012

  • Bryant, Zev; Oberstrass, Florian C.; Basu, Aakash
  • Current Opinion in Structural Biology, Vol. 22, Issue 3
  • DOI: 10.1016/j.sbi.2012.04.007

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA
journal, April 2012

  • Oberstrass, F. C.; Fernandes, L. E.; Bryant, Z.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 16
  • DOI: 10.1073/pnas.1113532109

Isopeptide bond in collagen- and fibrinogen-binding MSCRAMMs
journal, January 2016


Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9
journal, September 2016

  • Singh, Digvijay; Sternberg, Samuel H.; Fei, Jingyi
  • Nature Communications, Vol. 7, Issue 1
  • DOI: 10.1038/ncomms12778

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures
journal, February 2013

  • Naughton, Catherine; Avlonitis, Nicolaos; Corless, Samuel
  • Nature Structural & Molecular Biology, Vol. 20, Issue 3
  • DOI: 10.1038/nsmb.2509

Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes
journal, May 2014

  • Szczelkun, Mark D.; Tikhomirova, Maria S.; Sinkunas, Tomas
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 27
  • DOI: 10.1073/pnas.1402597111

A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9
journal, August 2017

  • Dagdas, Yavuz S.; Chen, Janice S.; Sternberg, Samuel H.
  • Science Advances, Vol. 3, Issue 8
  • DOI: 10.1126/sciadv.aao0027

Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension
journal, February 2014

  • Lebel, Paul; Basu, Aakash; Oberstrass, Florian C.
  • Nature Methods, Vol. 11, Issue 4
  • DOI: 10.1038/nmeth.2854

Intrinsic Rates and Activation Free Energies from Single-Molecule Pulling Experiments
journal, March 2006


Torque Spectroscopy of DNA: Base-Pair Stability, Boundary Effects, Backbending, and Breathing Dynamics
journal, April 2013


Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System
journal, June 2017


Understanding the Molecular Mechanisms of the CRISPR Toolbox Using Single Molecule Approaches
journal, January 2018


DNA stretching induces Cas9 off-target activity
journal, February 2019

  • Newton, Matthew D.; Taylor, Benjamin J.; Driessen, Rosalie P. C.
  • Nature Structural & Molecular Biology, Vol. 26, Issue 3
  • DOI: 10.1038/s41594-019-0188-z

ATP binding controls distinct structural transitions of Escherichia coli DNA gyrase in complex with DNA
journal, April 2012

  • Basu, Aakash; Schoeffler, Allyn J.; Berger, James M.
  • Nature Structural & Molecular Biology, Vol. 19, Issue 5
  • DOI: 10.1038/nsmb.2278

A Cas9-guide RNA complex preorganized for target DNA recognition
journal, June 2015


RNA-guided genetic silencing systems in bacteria and archaea
journal, February 2012

  • Wiedenheft, Blake; Sternberg, Samuel H.; Doudna, Jennifer A.
  • Nature, Vol. 482, Issue 7385, p. 331-338
  • DOI: 10.1038/nature10886

Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain
journal, May 2018

  • Sung, Keewon; Park, Jinho; Kim, Younggyu
  • Journal of the American Chemical Society, Vol. 140, Issue 25
  • DOI: 10.1021/jacs.8b03102

The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a
journal, February 2019

  • van Aelst, Kara; Martínez-Santiago, Carlos; Cross, Stephen
  • Genes, Vol. 10, Issue 2
  • DOI: 10.3390/genes10020169

A decade of discovery: CRISPR functions and applications
journal, June 2017


Transcription-dependent dynamic supercoiling is a short-range genomic force
journal, February 2013

  • Kouzine, Fedor; Gupta, Ashutosh; Baranello, Laura
  • Nature Structural & Molecular Biology, Vol. 20, Issue 3
  • DOI: 10.1038/nsmb.2517

GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases
journal, December 2014

  • Tsai, Shengdar Q.; Zheng, Zongli; Nguyen, Nhu T.
  • Nature Biotechnology, Vol. 33, Issue 2
  • DOI: 10.1038/nbt.3117

Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis
journal, April 2018

  • Singh, Digvijay; Wang, Yanbo; Mallon, John
  • Nature Structural & Molecular Biology, Vol. 25, Issue 4
  • DOI: 10.1038/s41594-018-0051-7

CRISPR-Cas immunity in prokaryotes
journal, October 2015


CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity
journal, April 2014


CRISPR-Cas guides the future of genetic engineering
journal, August 2018


Dynamic coupling between conformations and nucleotide states in DNA gyrase
journal, April 2018


DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity
journal, January 2018