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 Laboratory (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 = {Mon Mar 02 00:00:00 EST 2020},
month = {Mon Mar 02 00:00:00 EST 2020}
}
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