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Unlocking loxP to Track Genome Editing In Vivo

The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor g...

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Detalles Bibliográficos
Autores principales: Gendron, William A. C., Rubin, Jeffrey D., Hansen, Michael J., Nace, Rebecca A., Simone, Brandon W., Ekker, Stephen C., Barry, Michael A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394901/
https://www.ncbi.nlm.nih.gov/pubmed/34440379
http://dx.doi.org/10.3390/genes12081204
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author Gendron, William A. C.
Rubin, Jeffrey D.
Hansen, Michael J.
Nace, Rebecca A.
Simone, Brandon W.
Ekker, Stephen C.
Barry, Michael A.
author_facet Gendron, William A. C.
Rubin, Jeffrey D.
Hansen, Michael J.
Nace, Rebecca A.
Simone, Brandon W.
Ekker, Stephen C.
Barry, Michael A.
author_sort Gendron, William A. C.
collection PubMed
description The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR “fingerprinting” approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.
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spelling pubmed-83949012021-08-28 Unlocking loxP to Track Genome Editing In Vivo Gendron, William A. C. Rubin, Jeffrey D. Hansen, Michael J. Nace, Rebecca A. Simone, Brandon W. Ekker, Stephen C. Barry, Michael A. Genes (Basel) Article The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR “fingerprinting” approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models. MDPI 2021-08-03 /pmc/articles/PMC8394901/ /pubmed/34440379 http://dx.doi.org/10.3390/genes12081204 Text en © 2021 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
Gendron, William A. C.
Rubin, Jeffrey D.
Hansen, Michael J.
Nace, Rebecca A.
Simone, Brandon W.
Ekker, Stephen C.
Barry, Michael A.
Unlocking loxP to Track Genome Editing In Vivo
title Unlocking loxP to Track Genome Editing In Vivo
title_full Unlocking loxP to Track Genome Editing In Vivo
title_fullStr Unlocking loxP to Track Genome Editing In Vivo
title_full_unstemmed Unlocking loxP to Track Genome Editing In Vivo
title_short Unlocking loxP to Track Genome Editing In Vivo
title_sort unlocking loxp to track genome editing in vivo
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8394901/
https://www.ncbi.nlm.nih.gov/pubmed/34440379
http://dx.doi.org/10.3390/genes12081204
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