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A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing

CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually p...

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Autores principales: Wettengel, Jochen M., Hansen-Palmus, Lea, Yusova, Sofiya, Rust, Lauren, Biswas, Sreya, Carson, Julien, Ryu, Junghyun, Bimber, Benjamin N., Hennebold, Jon D., Burwitz, Benjamin J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10179647/
https://www.ncbi.nlm.nih.gov/pubmed/37175977
http://dx.doi.org/10.3390/ijms24098271
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author Wettengel, Jochen M.
Hansen-Palmus, Lea
Yusova, Sofiya
Rust, Lauren
Biswas, Sreya
Carson, Julien
Ryu, Junghyun
Bimber, Benjamin N.
Hennebold, Jon D.
Burwitz, Benjamin J.
author_facet Wettengel, Jochen M.
Hansen-Palmus, Lea
Yusova, Sofiya
Rust, Lauren
Biswas, Sreya
Carson, Julien
Ryu, Junghyun
Bimber, Benjamin N.
Hennebold, Jon D.
Burwitz, Benjamin J.
author_sort Wettengel, Jochen M.
collection PubMed
description CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.
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spelling pubmed-101796472023-05-13 A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing Wettengel, Jochen M. Hansen-Palmus, Lea Yusova, Sofiya Rust, Lauren Biswas, Sreya Carson, Julien Ryu, Junghyun Bimber, Benjamin N. Hennebold, Jon D. Burwitz, Benjamin J. Int J Mol Sci Article CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo. MDPI 2023-05-05 /pmc/articles/PMC10179647/ /pubmed/37175977 http://dx.doi.org/10.3390/ijms24098271 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Wettengel, Jochen M.
Hansen-Palmus, Lea
Yusova, Sofiya
Rust, Lauren
Biswas, Sreya
Carson, Julien
Ryu, Junghyun
Bimber, Benjamin N.
Hennebold, Jon D.
Burwitz, Benjamin J.
A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing
title A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing
title_full A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing
title_fullStr A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing
title_full_unstemmed A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing
title_short A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing
title_sort multifunctional and highly adaptable reporter system for crispr/cas editing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10179647/
https://www.ncbi.nlm.nih.gov/pubmed/37175977
http://dx.doi.org/10.3390/ijms24098271
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