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Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing

The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an ex...

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Autores principales: Lainšček, Duško, Forstnerič, Vida, Mikolič, Veronika, Malenšek, Špela, Pečan, Peter, Benčina, Mojca, Sever, Matjaž, Podgornik, Helena, Jerala, Roman
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9226073/
https://www.ncbi.nlm.nih.gov/pubmed/35739111
http://dx.doi.org/10.1038/s41467-022-31386-1
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author Lainšček, Duško
Forstnerič, Vida
Mikolič, Veronika
Malenšek, Špela
Pečan, Peter
Benčina, Mojca
Sever, Matjaž
Podgornik, Helena
Jerala, Roman
author_facet Lainšček, Duško
Forstnerič, Vida
Mikolič, Veronika
Malenšek, Špela
Pečan, Peter
Benčina, Mojca
Sever, Matjaž
Podgornik, Helena
Jerala, Roman
author_sort Lainšček, Duško
collection PubMed
description The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology.
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spelling pubmed-92260732022-06-25 Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing Lainšček, Duško Forstnerič, Vida Mikolič, Veronika Malenšek, Špela Pečan, Peter Benčina, Mojca Sever, Matjaž Podgornik, Helena Jerala, Roman Nat Commun Article The CRISPR/Cas system has emerged as a powerful and versatile genome engineering tool, revolutionizing biological and biomedical sciences, where an improvement of efficiency could have a strong impact. Here we present a strategy to enhance gene editing based on the concerted action of Cas9 and an exonuclease. Non-covalent recruitment of exonuclease to Cas9/gRNA complex via genetically encoded coiled-coil based domains, termed CCExo, recruited the exonuclease to the cleavage site and robustly increased gene knock-out due to progressive DNA strand recession at the cleavage site, causing decreased re-ligation of the nonedited DNA. CCExo exhibited increased deletion size and enhanced gene inactivation efficiency in the context of several DNA targets, gRNA selection, Cas variants, tested cell lines and type of delivery. Targeting a sequence-specific oncogenic chromosomal translocation using CCExo in cells of chronic myelogenous leukemia patients and in an animal model led to the reduction or elimination of cancer, establishing it as a highly specific tool for treating CML and potentially other appropriate diseases with genetic etiology. Nature Publishing Group UK 2022-06-23 /pmc/articles/PMC9226073/ /pubmed/35739111 http://dx.doi.org/10.1038/s41467-022-31386-1 Text en © The Author(s) 2022 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
Lainšček, Duško
Forstnerič, Vida
Mikolič, Veronika
Malenšek, Špela
Pečan, Peter
Benčina, Mojca
Sever, Matjaž
Podgornik, Helena
Jerala, Roman
Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing
title Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing
title_full Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing
title_fullStr Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing
title_full_unstemmed Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing
title_short Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing
title_sort coiled-coil heterodimer-based recruitment of an exonuclease to crispr/cas for enhanced gene editing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9226073/
https://www.ncbi.nlm.nih.gov/pubmed/35739111
http://dx.doi.org/10.1038/s41467-022-31386-1
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