Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice
| Autor: | Helen McLaughlin, Tess Torregrosa, Galina Marsh, Kathryn Koszka, Eric Marshall, Sam Hana, Chris E Henderson, Shanqin Xu, Michael Peterson, Chao Sun, Shih-Ching Lo, Olivia McKissick, Alexander Sorets, Michael Craft, Attila J. Fabian |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Central Nervous System
Molecular biology Article Transduction (genetics) Gene Knockout Techniques Mice In vivo Genetic model Genetics medicine CRISPR Animals Gene knockout Gene Editing Neurons biology Cas9 Cellular neuroscience Cell biology medicine.anatomical_structure nervous system biology.protein Molecular Medicine Neuron NeuN CRISPR-Cas Systems RNA Guide Kinetoplastida |
| Zdroj: | Gene Therapy |
| ISSN: | 1476-5462 0969-7128 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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