title: A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons.
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.
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Williams
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Catherine J
authors.initials: CJ
authors.lastname: Fricano-Kugler
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Getz
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Patrick D
authors.initials: PD
authors.lastname: Skelton
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Jeonghoon
authors.initials: J
authors.lastname: Lee
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Christian P
authors.initials: CP
authors.lastname: Rizzuto
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Geller
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Meijie
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
authors.forename: Bryan W
authors.initials: BW
authors.lastname: Luikart
mesh_terms: D000818:Animals; D001321:Autistic Disorder / Q000235:genetics* / Q000473:pathology; D064113:CRISPR-Cas Systems; D049108:Cell Enlargement; D005260:Female; D000072669:Gene Editing / Q000379:methods; D006801:Humans; D000074201:Katanin / Q000235:genetics; D008297:Male; D008810:Mice, Inbred C57BL; D009154:Mutation; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D051059:PTEN Phosphohydrolase / Q000235:genetics; D010641:Phenotype; D034622:RNA Interference; D012190:Retroviridae / Q000235:genetics*
chemical_list: D051059:PTEN Phosphohydrolase; C000607910:KATNAL2 protein, mouse; D000074201:Katanin