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PAM-flexible genome editing with an engineered chimeric Cas9
CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > N...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10550912/ https://www.ncbi.nlm.nih.gov/pubmed/37794046 http://dx.doi.org/10.1038/s41467-023-41829-y |
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author | Zhao, Lin Koseki, Sabrina R. T. Silverstein, Rachel A. Amrani, Nadia Peng, Christina Kramme, Christian Savic, Natasha Pacesa, Martin Rodríguez, Tomás C. Stan, Teodora Tysinger, Emma Hong, Lauren Yudistyra, Vivian Ponnapati, Manvitha R. Jacobson, Joseph M. Church, George M. Jakimo, Noah Truant, Ray Jinek, Martin Kleinstiver, Benjamin P. Sontheimer, Erik J. Chatterjee, Pranam |
author_facet | Zhao, Lin Koseki, Sabrina R. T. Silverstein, Rachel A. Amrani, Nadia Peng, Christina Kramme, Christian Savic, Natasha Pacesa, Martin Rodríguez, Tomás C. Stan, Teodora Tysinger, Emma Hong, Lauren Yudistyra, Vivian Ponnapati, Manvitha R. Jacobson, Joseph M. Church, George M. Jakimo, Noah Truant, Ray Jinek, Martin Kleinstiver, Benjamin P. Sontheimer, Erik J. Chatterjee, Pranam |
author_sort | Zhao, Lin |
collection | PubMed |
description | CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning. |
format | Online Article Text |
id | pubmed-10550912 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-105509122023-10-06 PAM-flexible genome editing with an engineered chimeric Cas9 Zhao, Lin Koseki, Sabrina R. T. Silverstein, Rachel A. Amrani, Nadia Peng, Christina Kramme, Christian Savic, Natasha Pacesa, Martin Rodríguez, Tomás C. Stan, Teodora Tysinger, Emma Hong, Lauren Yudistyra, Vivian Ponnapati, Manvitha R. Jacobson, Joseph M. Church, George M. Jakimo, Noah Truant, Ray Jinek, Martin Kleinstiver, Benjamin P. Sontheimer, Erik J. Chatterjee, Pranam Nat Commun Article CRISPR enzymes require a defined protospacer adjacent motif (PAM) flanking a guide RNA-programmed target site, limiting their sequence accessibility for robust genome editing applications. In this study, we recombine the PAM-interacting domain of SpRY, a broad-targeting Cas9 possessing an NRN > NYN (R = A or G, Y = C or T) PAM preference, with the N-terminus of Sc + +, a Cas9 with simultaneously broad, efficient, and accurate NNG editing capabilities, to generate a chimeric enzyme with highly flexible PAM preference: SpRYc. We demonstrate that SpRYc leverages properties of both enzymes to specifically edit diverse PAMs and disease-related loci for potential therapeutic applications. In total, the approaches to generate SpRYc, coupled with its robust flexibility, highlight the power of integrative protein design for Cas9 engineering and motivate downstream editing applications that require precise genomic positioning. Nature Publishing Group UK 2023-10-04 /pmc/articles/PMC10550912/ /pubmed/37794046 http://dx.doi.org/10.1038/s41467-023-41829-y 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 Zhao, Lin Koseki, Sabrina R. T. Silverstein, Rachel A. Amrani, Nadia Peng, Christina Kramme, Christian Savic, Natasha Pacesa, Martin Rodríguez, Tomás C. Stan, Teodora Tysinger, Emma Hong, Lauren Yudistyra, Vivian Ponnapati, Manvitha R. Jacobson, Joseph M. Church, George M. Jakimo, Noah Truant, Ray Jinek, Martin Kleinstiver, Benjamin P. Sontheimer, Erik J. Chatterjee, Pranam PAM-flexible genome editing with an engineered chimeric Cas9 |
title | PAM-flexible genome editing with an engineered chimeric Cas9 |
title_full | PAM-flexible genome editing with an engineered chimeric Cas9 |
title_fullStr | PAM-flexible genome editing with an engineered chimeric Cas9 |
title_full_unstemmed | PAM-flexible genome editing with an engineered chimeric Cas9 |
title_short | PAM-flexible genome editing with an engineered chimeric Cas9 |
title_sort | pam-flexible genome editing with an engineered chimeric cas9 |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10550912/ https://www.ncbi.nlm.nih.gov/pubmed/37794046 http://dx.doi.org/10.1038/s41467-023-41829-y |
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