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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...

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Autores principales: Sansbury, Brett M., Wagner, Amanda M., Nitzan, Erez, Tarcic, Gabi, Kmiec, Eric B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Mary Ann Liebert, Inc. 2018
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.
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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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