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Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases
Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and...
Autores principales: | , , , , , , , , , , , , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8271026/ https://www.ncbi.nlm.nih.gov/pubmed/34244505 http://dx.doi.org/10.1038/s41467-021-24454-5 |
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author | Schmidt, Moritz J. Gupta, Ashish Bednarski, Christien Gehrig-Giannini, Stefanie Richter, Florian Pitzler, Christian Gamalinda, Michael Galonska, Christina Takeuchi, Ryo Wang, Kui Reiss, Caroline Dehne, Kerstin Lukason, Michael J. Noma, Akiko Park-Windhol, Cindy Allocca, Mariacarmela Kantardzhieva, Albena Sane, Shailendra Kosakowska, Karolina Cafferty, Brian Tebbe, Jan Spencer, Sarah J. Munzer, Scott Cheng, Christopher J. Scaria, Abraham Scharenberg, Andrew M. Cohnen, André Coco, Wayne M. |
author_facet | Schmidt, Moritz J. Gupta, Ashish Bednarski, Christien Gehrig-Giannini, Stefanie Richter, Florian Pitzler, Christian Gamalinda, Michael Galonska, Christina Takeuchi, Ryo Wang, Kui Reiss, Caroline Dehne, Kerstin Lukason, Michael J. Noma, Akiko Park-Windhol, Cindy Allocca, Mariacarmela Kantardzhieva, Albena Sane, Shailendra Kosakowska, Karolina Cafferty, Brian Tebbe, Jan Spencer, Sarah J. Munzer, Scott Cheng, Christopher J. Scaria, Abraham Scharenberg, Andrew M. Cohnen, André Coco, Wayne M. |
author_sort | Schmidt, Moritz J. |
collection | PubMed |
description | Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a “GG” dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases. |
format | Online Article Text |
id | pubmed-8271026 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-82710262021-07-23 Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases Schmidt, Moritz J. Gupta, Ashish Bednarski, Christien Gehrig-Giannini, Stefanie Richter, Florian Pitzler, Christian Gamalinda, Michael Galonska, Christina Takeuchi, Ryo Wang, Kui Reiss, Caroline Dehne, Kerstin Lukason, Michael J. Noma, Akiko Park-Windhol, Cindy Allocca, Mariacarmela Kantardzhieva, Albena Sane, Shailendra Kosakowska, Karolina Cafferty, Brian Tebbe, Jan Spencer, Sarah J. Munzer, Scott Cheng, Christopher J. Scaria, Abraham Scharenberg, Andrew M. Cohnen, André Coco, Wayne M. Nat Commun Article Streptococcus pyogenes (Spy) Cas9 has potential as a component of gene therapeutics for incurable diseases. One of its limitations is its large size, which impedes its formulation and delivery in therapeutic applications. Smaller Cas9s are an alternative, but lack robust activity or specificity and frequently recognize longer PAMs. Here, we investigated four uncharacterized, smaller Cas9s and found three employing a “GG” dinucleotide PAM similar to SpyCas9. Protein engineering generated synthetic RNA-guided nucleases (sRGNs) with editing efficiencies and specificities exceeding even SpyCas9 in vitro and in human cell lines on disease-relevant targets. sRGN mRNA lipid nanoparticles displayed manufacturing advantages and high in vivo editing efficiency in the mouse liver. Finally, sRGNs, but not SpyCas9, could be packaged into all-in-one AAV particles with a gRNA and effected robust in vivo editing of non-human primate (NHP) retina photoreceptors. Human gene therapy efforts are expected to benefit from these improved alternatives to existing CRISPR nucleases. Nature Publishing Group UK 2021-07-09 /pmc/articles/PMC8271026/ /pubmed/34244505 http://dx.doi.org/10.1038/s41467-021-24454-5 Text en © The Author(s) 2021 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 Schmidt, Moritz J. Gupta, Ashish Bednarski, Christien Gehrig-Giannini, Stefanie Richter, Florian Pitzler, Christian Gamalinda, Michael Galonska, Christina Takeuchi, Ryo Wang, Kui Reiss, Caroline Dehne, Kerstin Lukason, Michael J. Noma, Akiko Park-Windhol, Cindy Allocca, Mariacarmela Kantardzhieva, Albena Sane, Shailendra Kosakowska, Karolina Cafferty, Brian Tebbe, Jan Spencer, Sarah J. Munzer, Scott Cheng, Christopher J. Scaria, Abraham Scharenberg, Andrew M. Cohnen, André Coco, Wayne M. Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases |
title | Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases |
title_full | Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases |
title_fullStr | Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases |
title_full_unstemmed | Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases |
title_short | Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases |
title_sort | improved crispr genome editing using small highly active and specific engineered rna-guided nucleases |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8271026/ https://www.ncbi.nlm.nih.gov/pubmed/34244505 http://dx.doi.org/10.1038/s41467-021-24454-5 |
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