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.

The LisH Domain-Containing N-Terminal Fragment is Important for the Localization, Dimerization, and Stability of Katnal2 in Tetrahymena

“…Detailed analyses of Xenopus embryos with depleted Katnal2 have revealed abnormalities during embryonic development and organogenesis, including a reduced brain size [7]. Defects in brain development are in agreement with observations in mice showing that Katnal2 also plays a role in neurons, specifically in dendrite arborization [10]. Interestingly, in humans, Katnal2 mutations may be associated with autism [11][12][13][14].…”

Fluorescent protein tagging of endogenous protein in brain neurons using CRISPR/Cas9-mediated knock-in and in utero electroporation techniques

“…NHEJ functions in both dividing and nondividing cells, whereas HDR functions only in dividing cells910. CRISPR/Cas9-mediated gene knock-out in postmitotic neurons in vivo has been reported such as mouse cortical neurons, mouse hippocampal dentate gyrus granule neurons, and mouse cerebellar Purkinje cells13232425262728. CRSIPR/Cas9-mediated gene knock-in has been shown in dividing cells, but not in postmitotic neurons.…”

CRISPR-ERA for Switching Off (Onco) Genes

“…Given the great potential of viral vectors in gene and cell therapy, five major classes of viral vectors-retroviruses [38], lentiviruses [39,40], adenoviruses [41,42], AAVs [43,44], and baculoviruses [45,46]-have been used to deliver CRISPR components into mammalian cells for targeted genome editing. The advantages and disadvantages of using these viral vectors for in vivo delivery of the CRISPR transgenes have been extensively reviewed [43,[47][48][49].…”