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Engineering designer beta cells with a CRISPR-Cas9 conjugation platform

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications th...

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Autores principales: Lim, Donghyun, Sreekanth, Vedagopuram, Cox, Kurt J., Law, Benjamin K., Wagner, Bridget K., Karp, Jeffrey M., Choudhary, Amit
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426819/
https://www.ncbi.nlm.nih.gov/pubmed/32792475
http://dx.doi.org/10.1038/s41467-020-17725-0
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author Lim, Donghyun
Sreekanth, Vedagopuram
Cox, Kurt J.
Law, Benjamin K.
Wagner, Bridget K.
Karp, Jeffrey M.
Choudhary, Amit
author_facet Lim, Donghyun
Sreekanth, Vedagopuram
Cox, Kurt J.
Law, Benjamin K.
Wagner, Bridget K.
Karp, Jeffrey M.
Choudhary, Amit
author_sort Lim, Donghyun
collection PubMed
description Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a β-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.
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spelling pubmed-74268192020-08-18 Engineering designer beta cells with a CRISPR-Cas9 conjugation platform Lim, Donghyun Sreekanth, Vedagopuram Cox, Kurt J. Law, Benjamin K. Wagner, Bridget K. Karp, Jeffrey M. Choudhary, Amit Nat Commun Article Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a β-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10. Nature Publishing Group UK 2020-08-13 /pmc/articles/PMC7426819/ /pubmed/32792475 http://dx.doi.org/10.1038/s41467-020-17725-0 Text en © The Author(s) 2020 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/.
spellingShingle Article
Lim, Donghyun
Sreekanth, Vedagopuram
Cox, Kurt J.
Law, Benjamin K.
Wagner, Bridget K.
Karp, Jeffrey M.
Choudhary, Amit
Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
title Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
title_full Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
title_fullStr Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
title_full_unstemmed Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
title_short Engineering designer beta cells with a CRISPR-Cas9 conjugation platform
title_sort engineering designer beta cells with a crispr-cas9 conjugation platform
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426819/
https://www.ncbi.nlm.nih.gov/pubmed/32792475
http://dx.doi.org/10.1038/s41467-020-17725-0
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