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

Journal Article · · Proceedings of the National Academy of Sciences of the United States of America
 [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

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.

Research Organization:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC); National Institutes of Health (NIH); Defense Advanced Research Projects Agency (DARPA); National Science Foundation (NSF)
Grant/Contract Number:
AC02-05CH11231
OSTI ID:
1633265
Journal Information:
Proceedings of the National Academy of Sciences of the United States of America, Vol. 117, Issue 11; ISSN 0027-8424
Publisher:
National Academy of SciencesCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 41 works
Citation information provided by
Web of Science

References (58)

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
DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 journal January 2014
High-throughput biochemical profiling reveals sequence determinants of dCas9 off-target binding and unbinding journal May 2017
Investigating DNA supercoiling in eukaryotic genomes journal April 2017
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
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
A transformation for the mechanical fingerprints of complex biomolecular interactions journal September 2013
Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection journal March 2015
An Information-Based Approach to Change-Point Analysis with Applications to Biophysics and Cell Biology journal July 2015
Transcription Under Torsion journal June 2013
Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease journal November 2016
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
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
Ten years of tension: single-molecule DNA mechanics journal January 2003
Cas9 as a versatile tool for engineering biology journal September 2013
Enhanced proofreading governs CRISPR–Cas9 targeting accuracy journal September 2017
Off-target Effects in CRISPR/Cas9-mediated Genome Engineering journal January 2015
Genomes in Focus: Development and Applications of CRISPR-Cas9 Imaging Technologies journal February 2018
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
A CRISPR–dCas Toolbox for Genetic Engineering and Synthetic Biology journal January 2019
Recent developments in single-molecule DNA mechanics journal June 2012
Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA journal April 2012
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
Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures journal February 2013
Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes journal May 2014
A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9 journal August 2017
Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension journal February 2014
Intrinsic Rates and Activation Free Energies from Single-Molecule Pulling Experiments journal March 2006
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
ATP binding controls distinct structural transitions of Escherichia coli DNA gyrase in complex with DNA journal April 2012
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
Target Specificity of Cas9 Nuclease via DNA Rearrangement Regulated by the REC2 Domain journal May 2018
The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a journal February 2019
A decade of discovery: CRISPR functions and applications journal June 2017
Transcription-dependent dynamic supercoiling is a short-range genomic force journal February 2013
GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases journal December 2014
Mechanisms of improved specificity of engineered Cas9s revealed by single-molecule FRET analysis journal April 2018
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
DNA supercoiling during transcription journal July 2016

Similar Records

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes
Journal Article · 2020 · Proceedings of the National Academy of Sciences of the United States of America · OSTI ID:1633267

A conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9
Journal Article · 2017 · Science Advances · OSTI ID:1625977

Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9
Journal Article · 2016 · Nature Communications · OSTI ID:1623849