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Zhang et al. Ageing Neur Dis 2023;3:24 https://dx.doi.org/10.20517/and.2023.18 Page 7 of 13

Monkey/Cynomolgus monkeys KO - Exons 6-12 (ANK, - Loss of neuronal cells - Impaired social interaction and apparent stereotypical [51]
+
PDZ)/mosaic - Increase of GFAP astrocytes locomotion
- All known SHANK3 protein - Altered expressions of postsynaptic receptors and scaffold - Delayed vocalization
isoforms reduced proteins - No obvious structural abnormality
- Lower glucose metabolism
- Fluoxetine can alleviate the behavioral deficits
Monkey/Cynomolgus monkeys KO - Exon 21 (PRO)/F0: mosaic; Not reported - Reduced overall sleep efficiency and muscle strength [52]
F1: heterozygous - Increased repetitive behaviors
- All known SHANK3 protein - Reduced exploration and social interaction
isoforms reduced - Fewer vocalizations
- Decreased grey matter
- Abnormal functional connectivity

AMPA: Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ANK: N-terminal ankyrin repeat domain; ASD: autism spectrum disorder; BDNF: brain-derived neurotrophic factor; KO: knock-out; LTP: long-
term potentiation; mEPSC: miniature excitatory postsynaptic current; mIPSC: miniature inhibitory postsynaptic current; mPFC: medial prefrontal cortex; NAC: nucleus accumbens; NeuN: neuronal nuclei;
NMDA: N-methyl-D-aspartate; NMJ: neuromuscular junction; NR1: NMDA receptor1; PDZ: postsynaptic density-95/discs large/zone occludens-1; PRO: a proline-rich region including homer and cortactin
binding sites; PSD: postsynaptic density; SAM: sterile alpha motif; SH3: Src homology domains; SHANK3: SH3 and multiple ankyrin repeat domains 3.

Targeted exons 6 and 12 of the monkey SHANK3 gene
[51]
In 2017, Zhao et al. generated the first SHANK3-edited monkey . Since a large deletion of shank3 in the mouse gene did not yield obvious phenotypes and
pathological changes, Zhao et al. aimed to establish a monkey model with a large SHANK3 gene deletion . They targeted two sites (exons 6 and 12) of the
[51]
monkey SHANK3 gene, which could completely disrupt all three longest SHANK3 isoforms in the fertilized eggs of cynomolgus monkeys. Although three
SHANK3 mutant monkeys were obtained, two (M1, M2) died before or right after birth, and one (M3) survived. A higher-than-expected embryonic and
perinatal lethality associated with SHANK3 targeting was observed, suggesting that SHANK3 is important for early development in primates. Analysis of

SHANK3-targeted monkeys revealed that the M1 monkey brains showed almost complete deletion of SHANK3 protein, accompanied by a dramatic reduction
in many postsynaptic receptors and scaffold proteins, such as GluN2B/mGluR5/PSD95. Meanwhile, the SHANK3-deficient monkey showed a significant loss
of NeuN+ neurons co-exhibited by an increase of GFAP+ astrocytes [Figure 1]. These findings provide strong evidence for neuronal loss resulting from

SHANK3 depletion, which has not been reported in any line of shank3-knockout mice. This demonstrates that SHANK3 plays a unique and critical role in
early brain development in primate brains.

For the living SHANK3-targeted monkey (M3), a longitudinal and repeated investigation over a 2-year period had been performed . This investigation
[57]
revealed that the SHANK3-targeted monkey developed the core features of behavioral phenotypes of ASD, including impaired social interaction, apparent
stereotypical locomotion, and delayed vocalization . Although the exact meaning of delayed vocalization is not clear, the defect in vocalization may resemble
[57]