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CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract
Extraordinary efforts are underway to offer greater versatility and broader applications for CRISPR-directed gene editing. Here, we report the establishment of a system for studying this process in a mammalian cell-free extract prepared from HEK-293 human embryonic kidney cells. A ribonucleoprotein...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Mary Ann Liebert, Inc.
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345151/ https://www.ncbi.nlm.nih.gov/pubmed/30687813 http://dx.doi.org/10.1089/crispr.2018.0006 |
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author | Sansbury, Brett M. Wagner, Amanda M. Nitzan, Erez Tarcic, Gabi Kmiec, Eric B. |
author_facet | Sansbury, Brett M. Wagner, Amanda M. Nitzan, Erez Tarcic, Gabi Kmiec, Eric B. |
author_sort | Sansbury, Brett M. |
collection | PubMed |
description | Extraordinary efforts are underway to offer greater versatility and broader applications for CRISPR-directed gene editing. Here, we report the establishment of a system for studying this process in a mammalian cell-free extract prepared from HEK-293 human embryonic kidney cells. A ribonucleoprotein (RNP) particle and a mammalian cell-free extract coupled with a genetic readout are used to generate and identify specific deletions or insertions within a plasmid target. A Cpf1 (Cas12a) RNP induces a double-stranded break, and the cell-free extract provides the appropriate enzymatic activities to direct specific deletion through resection and homology directed repair in the presence of single- and double-stranded donor DNA. This cell-free system establishes a foundation to study the heterogeneous products of gene editing, as well as the relationship between nonhomologous end joining and homology directed repair and related regulatory circuitries simultaneously in a controlled environment. |
format | Online Article Text |
id | pubmed-6345151 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Mary Ann Liebert, Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-63451512019-01-24 CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract Sansbury, Brett M. Wagner, Amanda M. Nitzan, Erez Tarcic, Gabi Kmiec, Eric B. CRISPR J Research Articles Extraordinary efforts are underway to offer greater versatility and broader applications for CRISPR-directed gene editing. Here, we report the establishment of a system for studying this process in a mammalian cell-free extract prepared from HEK-293 human embryonic kidney cells. A ribonucleoprotein (RNP) particle and a mammalian cell-free extract coupled with a genetic readout are used to generate and identify specific deletions or insertions within a plasmid target. A Cpf1 (Cas12a) RNP induces a double-stranded break, and the cell-free extract provides the appropriate enzymatic activities to direct specific deletion through resection and homology directed repair in the presence of single- and double-stranded donor DNA. This cell-free system establishes a foundation to study the heterogeneous products of gene editing, as well as the relationship between nonhomologous end joining and homology directed repair and related regulatory circuitries simultaneously in a controlled environment. Mary Ann Liebert, Inc. 2018-04-01 2018-04-01 /pmc/articles/PMC6345151/ /pubmed/30687813 http://dx.doi.org/10.1089/crispr.2018.0006 Text en © Brett M. Sansbury et al. 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are cited. |
spellingShingle | Research Articles Sansbury, Brett M. Wagner, Amanda M. Nitzan, Erez Tarcic, Gabi Kmiec, Eric B. CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract |
title | CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract |
title_full | CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract |
title_fullStr | CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract |
title_full_unstemmed | CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract |
title_short | CRISPR-Directed In Vitro Gene Editing of Plasmid DNA Catalyzed by Cpf1 (Cas12a) Nuclease and a Mammalian Cell-Free Extract |
title_sort | crispr-directed in vitro gene editing of plasmid dna catalyzed by cpf1 (cas12a) nuclease and a mammalian cell-free extract |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345151/ https://www.ncbi.nlm.nih.gov/pubmed/30687813 http://dx.doi.org/10.1089/crispr.2018.0006 |
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