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 »
- Authors:
-
- Stanford University, Stanford, CA (United States)
- Univ. of California, Berkeley, CA (United States)
- 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. doi: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}
}
Free Publicly Available Full Text
Publisher's Version of Record
Other availability
Search WorldCat to find libraries that may hold this journal
Cited by: 2 works
Citation information provided by
Web of Science
Web of Science
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.
Works referenced in this record:
Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage
journal, January 2016
- Jiang, F.; Taylor, D. W.; Chen, J. S.
- Science, Vol. 351, Issue 6275
The new frontier of genome engineering with CRISPR-Cas9
journal, November 2014
- Doudna, Jennifer A.; Charpentier, Emmanuelle
- Science, Vol. 346, Issue 6213
Genome Editing B.C. (Before CRISPR): Lasting Lessons from the “Old Testament”
journal, February 2018
- Urnov, Fyodor D.
- The CRISPR Journal, Vol. 1, Issue 1
Conformational control of DNA target cleavage by CRISPR–Cas9
journal, October 2015
- Sternberg, Samuel H.; LaFrance, Benjamin; Kaplan, Matias
- Nature, Vol. 527, Issue 7576
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9
journal, January 2014
- Sternberg, Samuel H.; Redding, Sy; Jinek, Martin
- Nature, Vol. 507, Issue 7490
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
Investigating DNA supercoiling in eukaryotic genomes
journal, April 2017
- Corless, Samuel; Gilbert, Nick
- Briefings in Functional Genomics, Vol. 16, Issue 6
CRISPR-Mediated Adaptive Immune Systems in Bacteria and Archaea
journal, June 2013
- Sorek, Rotem; Lawrence, C. Martin; Wiedenheft, Blake
- Annual Review of Biochemistry, Vol. 82, Issue 1
CRISPR/Cas9 in Genome Editing and Beyond
journal, June 2016
- Wang, Haifeng; La Russa, Marie; Qi, Lei S.
- Annual Review of Biochemistry, Vol. 85, Issue 1
Kinetics of dCas9 target search in Escherichia coli
journal, September 2017
- Jones, Daniel Lawson; Leroy, Prune; Unoson, Cecilia
- Science, Vol. 357, Issue 6358
Structural transitions and elasticity from torque measurements on DNA
journal, July 2003
- Bryant, Zev; Stone, Michael D.; Gore, Jeff
- Nature, Vol. 424, Issue 6946
Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: Analogous measurements in insect and human cells
journal, October 1980
- Sinden, R.
- Cell, Vol. 21, Issue 3
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
Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection
journal, March 2015
- Rutkauskas, Marius; Sinkunas, Tomas; Songailiene, Inga
- Cell Reports, Vol. 10, Issue 9
An Information-Based Approach to Change-Point Analysis with Applications to Biophysics and Cell Biology
journal, July 2015
- Wiggins, Paul A.
- Biophysical Journal, Vol. 109, Issue 2
Transcription Under Torsion
journal, June 2013
- Ma, Jie; Bai, Lu; Wang, Michelle D.
- Science, Vol. 340, Issue 6140
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
The effect of force on thermodynamics and kinetics of single molecule reactions
journal, December 2002
- Tinoco Jr, Ignacio; Bustamante, Carlos
- Biophysical Chemistry, Vol. 101-102
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
Development and Applications of CRISPR-Cas9 for Genome Engineering
journal, June 2014
- Hsu, Patrick D.; Lander, Eric S.; Zhang, Feng
- Cell, Vol. 157, Issue 6
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
Ten years of tension: single-molecule DNA mechanics
journal, January 2003
- Bustamante, Carlos; Bryant, Zev; Smith, Steven B.
- Nature, Vol. 421, Issue 6921
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
Enhanced proofreading governs CRISPR–Cas9 targeting accuracy
journal, September 2017
- Chen, Janice S.; Dagdas, Yavuz S.; Kleinstiver, Benjamin P.
- Nature, Vol. 550, Issue 7676
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
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
The Development of an Information Criterion for Change-Point Analysis
journal, March 2016
- LaMont, Colin H.; Wiggins, Paul A.
- Neural Computation, Vol. 28, Issue 3
Multimodal Measurements of Single-Molecule Dynamics Using FluoRBT
journal, January 2018
- Ivanov, Ivan E.; Lebel, Paul; Oberstrass, Florian C.
- Biophysical Journal, Vol. 114, Issue 2
A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012
- Jinek, M.; Chylinski, K.; Fonfara, I.
- Science, Vol. 337, Issue 6096, p. 816-821
Cellular roles of DNA topoisomerases: a molecular perspective
journal, June 2002
- Wang, James C.
- Nature Reviews Molecular Cell Biology, Vol. 3, Issue 6
A CRISPR–dCas Toolbox for Genetic Engineering and Synthetic Biology
journal, January 2019
- Xu, Xiaoshu; Qi, Lei S.
- Journal of Molecular Biology, Vol. 431, Issue 1
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
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
Isopeptide bond in collagen- and fibrinogen-binding MSCRAMMs
journal, January 2016
- Sridharan, Upasana; Ponnuraj, Karthe
- Biophysical Reviews, Vol. 8, Issue 1
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
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
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
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
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
Intrinsic Rates and Activation Free Energies from Single-Molecule Pulling Experiments
journal, March 2006
- Dudko, Olga; Hummer, Gerhard; Szabo, Attila
- Physical Review Letters, Vol. 96, Issue 10
Torque Spectroscopy of DNA: Base-Pair Stability, Boundary Effects, Backbending, and Breathing Dynamics
journal, April 2013
- Oberstrass, Florian C.; Fernandes, Louis E.; Lebel, Paul
- Physical Review Letters, Vol. 110, Issue 17
Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System
journal, June 2017
- Xiao, Yibei; Luo, Min; Hayes, Robert P.
- Cell, Vol. 170, Issue 1
Understanding the Molecular Mechanisms of the CRISPR Toolbox Using Single Molecule Approaches
journal, January 2018
- Singh, Digvijay; Ha, Taekjip
- ACS Chemical Biology, Vol. 13, Issue 3
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
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
A Cas9-guide RNA complex preorganized for target DNA recognition
journal, June 2015
- Jiang, F.; Zhou, K.; Ma, L.
- Science, Vol. 348, Issue 6242
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
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
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
A decade of discovery: CRISPR functions and applications
journal, June 2017
- Barrangou, Rodolphe; Horvath, Philippe
- Nature Microbiology, Vol. 2, Issue 7
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
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
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
CRISPR-Cas immunity in prokaryotes
journal, October 2015
- Marraffini, Luciano A.
- Nature, Vol. 526, Issue 7571
CRISPR-Cas Systems: Prokaryotes Upgrade to Adaptive Immunity
journal, April 2014
- Barrangou, Rodolphe; Marraffini, Luciano A.
- Molecular Cell, Vol. 54, Issue 2
CRISPR-Cas guides the future of genetic engineering
journal, August 2018
- Knott, Gavin J.; Doudna, Jennifer A.
- Science, Vol. 361, Issue 6405
Dynamic coupling between conformations and nucleotide states in DNA gyrase
journal, April 2018
- Basu, Aakash; Hobson, Matthew; Lebel, Paul
- Nature Chemical Biology, Vol. 14, Issue 6
DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity
journal, January 2018
- Gong, Shanzhong; Yu, Helen Hong; Johnson, Kenneth A.
- Cell Reports, Vol. 22, Issue 2