A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons

Retroviruses expressing a fluorescent protein, Cas9, and a small guide RNA are used to mimic nonsense PTEN mutations from autism patients in developing mouse neurons. We compare the cellular phenotype elicited by CRISPR-Cas9 to those elicited using shRNA or Cre/Lox technologies and find that knockdo...

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Bibliographic Details
Published in:Scientific reports 2016-05, Vol.6 (1), p.25611-25611, Article 25611
Main Authors: Williams, Michael R, Fricano-Kugler, Catherine J, Getz, Stephanie A, Skelton, Patrick D, Lee, Jeonghoon, Rizzuto, Christian P, Geller, Joseph S, Li, Meijie, Luikart, Bryan W
Format: Article
Language:English
Subjects:
Animals
Autism
Autistic Disorder - genetics
Autistic Disorder - pathology
Cell Enlargement
Clonal deletion
CRISPR
CRISPR-Cas Systems
Dendritic branching
Female
Gene deletion
Gene Editing - methods
Genotype & phenotype
Humans
Hypertrophy
Katanin - genetics
Male
Mice, Inbred C57BL
Mutation
Neurons
Neurons - metabolism
Neurons - pathology
Phenotype
PTEN Phosphohydrolase - genetics
PTEN protein
Retroviridae - genetics
Ribonucleic acid
RNA
RNA Interference
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Summary:Retroviruses expressing a fluorescent protein, Cas9, and a small guide RNA are used to mimic nonsense PTEN mutations from autism patients in developing mouse neurons. We compare the cellular phenotype elicited by CRISPR-Cas9 to those elicited using shRNA or Cre/Lox technologies and find that knockdown or knockout (KO) produced a corresponding moderate or severe neuronal hypertrophy in all cells. In contrast, the Cas9 approach produced missense and nonsense Pten mutations, resulting in a mix of KO-equivalent hypertrophic and wild type-like phenotypes. Importantly, despite this mixed phenotype, the neuronal hypertrophy resulting from Pten loss was evident on average in the population of manipulated cells. Having reproduced the known Pten KO phenotype using the CRISPR-Cas9 system we design viruses to target a gene that has recently been associated with autism, KATNAL2. Katnal2 deletion in the mouse results in decreased dendritic arborization of developing neurons. We conclude that retroviral implementation of the CRISPR-Cas9 system is an efficient system for cellular phenotype discovery in wild-type animals.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep25611