CRISPR-Cas9 Mediated SHANK3 Gene Editing in Macaques Reveals Autism-Like Behaviors and Brain Connectivity Abnormalities

The SHANK3 gene encodes a scaffolding protein on excitatory synapses. It plays a crucial role in organizing other proteins on the postsynaptic cell membrane, coordinating the response of cells to signals from presynaptic cells. Mutations in this gene have been linked to various diseases, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome. Studies have shown that even single mutations in SHANK3 can lead to these conditions, with over 1% of congenital ASD cases attributed to SHANK3 gene mutations.

Rodent models have proven useful in understanding the role of SHANK3 in ASD, but their limitations in mimicking human-like social behavior and cognitive complexity warrant the development of more complex animal models. Macaques, with their brain structures and cognitive abilities closer to humans, offer a valuable alternative.

Leveraging CRISPR-Cas9 technology, Professor Desimone's research team successfully edited the SHANK3 gene in macaques, creating a line of SHANK3 mutant macaques. They further generated first-generation offspring carrying the SHANK3 mutation through breeding with the mutant males. Genotype analysis and brain biopsies confirmed the presence of the SHANK3 mutation and a corresponding decrease in SHANK3 protein levels. This confirmed the effectiveness of CRISPR gene editing in macaques and its capacity for transmission across generations.

The research team then investigated whether SHANK3 mutant macaques exhibited autism-related behaviors. Behavioral assessments revealed significant differences between the mutant and control macaques. The mutant macaques showed reduced overall activity levels, prolonged sleep onset times, and frequent awakenings during sleep, indicating a compromised sleep efficiency. Additionally, the mutant macaques exhibited an increase in repetitive behaviors, including backflips, finger licking, and cage biting, although these behaviors displayed individual variations. These macaques also showed reduced exploration of their habitat, decreased vocalizations, and fewer social interactions.

Eye-tracking analysis during video presentations further revealed abnormal eye movement patterns in the mutant macaques. Pupil reflexes were delayed, video viewing times were prolonged, lip-smacking behavior was increased, and gaze duration was shortened. These behavioral abnormalities strongly resemble some of the characteristics observed in individuals with ASD or Phelan-McDermid syndrome.

To investigate the potential neuroanatomical and functional connectivity differences in the brains of SHANK3 mutant macaques, the research team conducted MRI scans. They discovered a reduction in gray matter volume in the brains of the mutant macaques, while white matter and cerebrospinal fluid volume remained unchanged. The MRI also revealed functional connectivity abnormalities in the mutant macaques. Long-range connections between certain brain regions, particularly those within the default mode network (including the posterior cingulate cortex, medial prefrontal cortex, and motor areas), were decreased. Local connectivity deficits were also observed in the thalamus and striatum. Conversely, the somatosensory cortex, extrastriate cortical areas, and posterior cingulate cortex showed increased local connectivity. These findings suggest that the brain structure and function of SHANK3 mutant macaques share similarities with those of humans with ASD.

This study, utilizing CRISPR-Cas9 technology, generated SHANK3 mutant macaques exhibiting autism-related behaviors and brain abnormalities. This model provides a valuable platform for further research on the complex mechanisms underlying ASD and for developing potential therapies.