Cas9 interrogates DNA in discrete steps modulated by mismatches and supercoiling
- Stanford University, Stanford, CA (United States)
- Univ. of California, Berkeley, CA (United States)
- 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
Web of Science
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