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Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation
Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is gene...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group US
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10643121/ https://www.ncbi.nlm.nih.gov/pubmed/37828409 http://dx.doi.org/10.1038/s41594-023-01104-6 |
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author | Wang, Jinglong Le Gall, Julien Frock, Richard L. Strick, Terence R. |
author_facet | Wang, Jinglong Le Gall, Julien Frock, Richard L. Strick, Terence R. |
author_sort | Wang, Jinglong |
collection | PubMed |
description | Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3′ overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR–SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends. |
format | Online Article Text |
id | pubmed-10643121 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group US |
record_format | MEDLINE/PubMed |
spelling | pubmed-106431212023-11-14 Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation Wang, Jinglong Le Gall, Julien Frock, Richard L. Strick, Terence R. Nat Struct Mol Biol Article Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3′ overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR–SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends. Nature Publishing Group US 2023-10-12 2023 /pmc/articles/PMC10643121/ /pubmed/37828409 http://dx.doi.org/10.1038/s41594-023-01104-6 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Wang, Jinglong Le Gall, Julien Frock, Richard L. Strick, Terence R. Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation |
title | Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation |
title_full | Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation |
title_fullStr | Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation |
title_full_unstemmed | Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation |
title_short | Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation |
title_sort | shifted pams generate dna overhangs and enhance spcas9 post-catalytic complex dissociation |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10643121/ https://www.ncbi.nlm.nih.gov/pubmed/37828409 http://dx.doi.org/10.1038/s41594-023-01104-6 |
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