Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice.
Journal
Gene therapy
ISSN: 1476-5462
Titre abrégé: Gene Ther
Pays: England
ID NLM: 9421525
Informations de publication
Date de publication:
11 2021
11 2021
Historique:
received:
14
07
2020
accepted:
15
01
2021
revised:
29
11
2020
pubmed:
10
2
2021
medline:
3
3
2022
entrez:
9
2
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 33558692
doi: 10.1038/s41434-021-00224-2
pii: 10.1038/s41434-021-00224-2
pmc: PMC8599009
doi:
Substances chimiques
RNA, Guide
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
646-658Informations de copyright
© 2021. The Author(s), under exclusive licence to Springer Nature Limited part of Springer Nature.
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