A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons
Document Type
Article
Publication Date
5-10-2016
Publication Title
Scientific Reports
Department
Geisel School of Medicine
Abstract
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.
DOI
10.1038/srep25611
Dartmouth Digital Commons Citation
Williams, Michael R.; Fricano-Kugler, Catherine J.; Getz, Stephanie A.; Skelton, Patrick D.; Lee, Jeonghoon; Rizzuto, Christian P.; Geller, Joseph S.; Li, Meijie; and Luikart, Bryan W., "A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons" (2016). Dartmouth Scholarship. 375.
https://digitalcommons.dartmouth.edu/facoa/375
