A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons
Michael R Williams1, Catherine J Fricano-Kugler1, Stephanie A Getz1
1Department of Physiology and Neurobiology, Geisel School of Medicine at Dartmouth College, USA.
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.
Insights
CRISPR-Cas9 efficiently mimics autism-related gene mutations in mouse neurons. This method aids in discovering cellular phenotypes, revealing neuronal hypertrophy from PTEN loss and altered dendritic structures from KATNAL2 deletion.
Area of Science:
- Neuroscience
- Genetics
- Molecular Biology
Background:
- PTEN mutations are linked to autism spectrum disorder (ASD).
- Understanding gene function in neuronal development is crucial for autism research.
- CRISPR-Cas9 offers a precise tool for genetic manipulation in cellular models.
Purpose of the Study:
- To evaluate CRISPR-Cas9's efficacy in modeling PTEN mutations in mouse neurons.
- To compare CRISPR-Cas9 with shRNA and Cre/Lox systems for phenotype discovery.
- To investigate the cellular effects of KATNAL2 deletion, a gene associated with autism.
Main Methods:
- Retroviruses encoding Cas9 and guide RNA were used to induce PTEN mutations in developing mouse neurons.
- CRISPR-Cas9 mediated mutations were compared to shRNA knockdown and Cre/Lox knockout models.
- Viral delivery of CRISPR-Cas9 was employed to delete KATNAL2 in mouse models.
Main Results:
- CRISPR-Cas9 successfully mimicked PTEN nonsense mutations, causing neuronal hypertrophy.
- Compared to shRNA/Cre/Lox, Cas9 generated a mix of hypertrophic and wild-type phenotypes due to missense/nonsense mutations.
- KATNAL2 deletion resulted in reduced dendritic arborization in developing neurons.
- Neuronal hypertrophy from PTEN loss was observed on average in manipulated cells.
Conclusions:
- Retroviral CRISPR-Cas9 is an effective system for discovering cellular phenotypes in vivo.
- This approach advances the study of autism-associated genes like PTEN and KATNAL2.
- The findings provide insights into the genetic basis of neurodevelopmental disorders.
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