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Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice
CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, th...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8599009/ https://www.ncbi.nlm.nih.gov/pubmed/33558692 http://dx.doi.org/10.1038/s41434-021-00224-2 |
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author | Hana, Sam Peterson, Michael McLaughlin, Helen Marshall, Eric Fabian, Attila J. McKissick, Olivia Koszka, Kathryn Marsh, Galina Craft, Michael Xu, Shanqin Sorets, Alexander Torregrosa, Tess Sun, Chao Henderson, Chris E. Lo, Shih-Ching |
author_facet | Hana, Sam Peterson, Michael McLaughlin, Helen Marshall, Eric Fabian, Attila J. McKissick, Olivia Koszka, Kathryn Marsh, Galina Craft, Michael Xu, Shanqin Sorets, Alexander Torregrosa, Tess Sun, Chao Henderson, Chris E. Lo, Shih-Ching |
author_sort | Hana, Sam |
collection | PubMed |
description | CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research. |
format | Online Article Text |
id | pubmed-8599009 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-85990092021-12-02 Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice Hana, Sam Peterson, Michael McLaughlin, Helen Marshall, Eric Fabian, Attila J. McKissick, Olivia Koszka, Kathryn Marsh, Galina Craft, Michael Xu, Shanqin Sorets, Alexander Torregrosa, Tess Sun, Chao Henderson, Chris E. Lo, Shih-Ching Gene Ther Article CRISPR-Cas systems have emerged as a powerful tool to generate genetic models for studying normal and diseased central nervous system (CNS). Targeted gene disruption at specific loci has been demonstrated successfully in non-dividing neurons. Despite its simplicity, high specificity and low cost, the efficiency of CRISPR-mediated knockout in vivo can be substantially impacted by many parameters. Here, we used CRISPR-Cas9 to disrupt the neuronal-specific gene, NeuN, and optimized key parameters to achieve effective gene knockout broadly in the CNS in postnatal mice. Three cell lines and two primary neuron cultures were used to validate the disruption of NeuN by single-guide RNAs (sgRNA) harboring distinct spacers and scaffold sequences. This triage identified an optimal sgRNA design with the highest NeuN disruption in in vitro and in vivo systems. To enhance CRISPR efficiency, AAV-PHP.B, a vector with superior neuronal transduction, was used to deliver this sgRNA in Cas9 mice via neonatal intracerebroventricular (ICV) injection. This approach resulted in 99.4% biallelic indels rate in the transduced cells, leading to greater than 70% reduction of total NeuN proteins in the cortex, hippocampus and spinal cord. This work contributes to the optimization of CRISPR-mediated knockout and will be beneficial for fundamental and preclinical research. Nature Publishing Group UK 2021-02-08 2021 /pmc/articles/PMC8599009/ /pubmed/33558692 http://dx.doi.org/10.1038/s41434-021-00224-2 Text en © The Author(s), under exclusive licence to Springer Nature Limited part of Springer Nature 2021 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . |
spellingShingle | Article Hana, Sam Peterson, Michael McLaughlin, Helen Marshall, Eric Fabian, Attila J. McKissick, Olivia Koszka, Kathryn Marsh, Galina Craft, Michael Xu, Shanqin Sorets, Alexander Torregrosa, Tess Sun, Chao Henderson, Chris E. Lo, Shih-Ching Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice |
title | Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice |
title_full | Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice |
title_fullStr | Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice |
title_full_unstemmed | Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice |
title_short | Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice |
title_sort | highly efficient neuronal gene knockout in vivo by crispr-cas9 via neonatal intracerebroventricular injection of aav in mice |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8599009/ https://www.ncbi.nlm.nih.gov/pubmed/33558692 http://dx.doi.org/10.1038/s41434-021-00224-2 |
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