Unlocking <i>lox</i>P 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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doaj-ee033836d72f4f77a671df618ef06c392021-08-26T13:46:56ZengMDPI AGGenes2073-44252021-08-01121204120410.3390/genes12081204Unlocking <i>lox</i>P to Track Genome Editing In VivoWilliam A. C. Gendron0Jeffrey D. Rubin1Michael J. Hansen2Rebecca A. Nace3Brandon W. Simone4Stephen C. Ekker5Michael A. Barry6Virology and Gene Therapy Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN 55905, USAVirology and Gene Therapy Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN 55905, USAVirology and Gene Therapy Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN 55905, USAVirology and Gene Therapy Graduate Program, Mayo Clinic Graduate School of Biomedical Sciences, Rochester, MN 55905, USADepartment of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USADepartment of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, USADepartment of Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN 55905, USAThe 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 <i>lox</i>P including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within <i>lox</i>P, 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 <i>lox</i>P animal models.https://www.mdpi.com/2073-4425/12/8/1204CRISPRSaCas9ErCas12a<i>lox</i>PCreluciferase reporter |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
William A. C. Gendron Jeffrey D. Rubin Michael J. Hansen Rebecca A. Nace Brandon W. Simone Stephen C. Ekker Michael A. Barry |
spellingShingle |
William A. C. Gendron Jeffrey D. Rubin Michael J. Hansen Rebecca A. Nace Brandon W. Simone Stephen C. Ekker Michael A. Barry Unlocking <i>lox</i>P to Track Genome Editing In Vivo Genes CRISPR SaCas9 ErCas12a <i>lox</i>P Cre luciferase reporter |
author_facet |
William A. C. Gendron Jeffrey D. Rubin Michael J. Hansen Rebecca A. Nace Brandon W. Simone Stephen C. Ekker Michael A. Barry |
author_sort |
William A. C. Gendron |
title |
Unlocking <i>lox</i>P to Track Genome Editing In Vivo |
title_short |
Unlocking <i>lox</i>P to Track Genome Editing In Vivo |
title_full |
Unlocking <i>lox</i>P to Track Genome Editing In Vivo |
title_fullStr |
Unlocking <i>lox</i>P to Track Genome Editing In Vivo |
title_full_unstemmed |
Unlocking <i>lox</i>P to Track Genome Editing In Vivo |
title_sort |
unlocking <i>lox</i>p to track genome editing in vivo |
publisher |
MDPI AG |
series |
Genes |
issn |
2073-4425 |
publishDate |
2021-08-01 |
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 <i>lox</i>P including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within <i>lox</i>P, 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 <i>lox</i>P animal models. |
topic |
CRISPR SaCas9 ErCas12a <i>lox</i>P Cre luciferase reporter |
url |
https://www.mdpi.com/2073-4425/12/8/1204 |
work_keys_str_mv |
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