Modernising autism spectrum disorder model engineering and treatment via CRISPR-Cas9: A gene reprogramming approach

Table 1 Summary of clustered regularly interspaced palindromic repeats-associated protein 9 engineered models of autism spectrum disorder

Ref.	Model	Gene mutation/syndrome	Observed alterations
Cellular
[32]	ES cells	SHANK3/Phelan-McDermid syndrome	Altered neuronal morphology and synaptic connectivity; impaired Ih channels
[33]	iPSCs	CNTN5 or EHMT2/idiopathic ASD	Increased synaptic excitatory neuron activity
[34]	iPSCs	EHMT1/Kleefstra syndrome	Upregulation of NMDAR1; neuronal network impairments
[35]	iPSCs	ADNP, DDX3X and FOXP1/idiopathic ASD	Increased neurogenesis
[36]	iPSCs	CHD8/idiopathic ASD	Dysregulated expression of genes associated with human brain volume or head size
[37]	iPSCs	PTCHD1-AS/idiopathic ASD	Impaired excitatory synaptic function (NMDAR hypofunction); synaptic impairment
[38]	iPSCs	COSMOC/idiopathic ASD	Destabilization of lipid and energy metabolism; affected neuronal maturation
Organoids
[41]	Cerebral	PTEN/idiopathic ASD, CHD8/idiopathic ASD, SUV420H1/ idiopathic ASD	Increased upper layer colossal neurons, cycling progenitor neuron; high outer radial glial cells; increased cortical interneurons; increased newly born deep layer projection neurons
[42]	Cerebral	RAB39b/ idiopathic ASD	Increased NPC proliferation
[44]	Cortical and neurons	MECP2/Rett syndrome	Dysregulation in genes of neuronal and glial cells
[46]	Cortical	UBE3A/Angelman syndrome	Dysfunction in big potassium channel dysfunction causing increased neuronal excitability
[47]	Cortical	TSC1 or 2/Tuberous sclerosis complex	Affected cortical neurons and glial cell development
[49]	Cortico-striatal organoids	SHANK3/ Phelan-McDermid syndrome	Enhanced neuronal excitability; dysregulated expression of protocadherins and zinc-finger genes
[50]	Cortical	FMR1/Fragile X syndrome	Increased number of glial cells and bigger organoid size
Animal Models
[52]	Cynomolgus macaques	SHANK3/ Phelan-McDermid syndrome	Sleep disturbances; increased repetitive behaviour, motor deficit; social and learning impairment; aberrant neural circuit connectivity
[53]	Mice	ARID1B/idiopathic ASD	Social behaviour impairment; altered vocalization; anxiety-like behaviour; neuroanatomical abnormalities; growth impairment
[54]	Mice	CHD8/idiopathic ASD	Cognitive impairment; disrupted pathways involved in neurogenesis, neuroimmune signalling, synaptic processes
[55]	Mice	ASH1L/idiopathic ASD	Dysregulated epigenetic modification; upregulation of neurexin-1α
[56]	Rat	CYFIP1/idiopathic ASD	Extensive changes in white matter; myelin sheath thinning in corpus callosum; abnormal oligodendrocytes; behavioural inflexibility
[57]	Rat	TCF4/idiopathic ASD	Attenuated action potential output; alteration in electrophysiological properties in neurons
[58]	Rat	UBE3A/idiopathic ASD	Deficits in motor coordination as well as learning and memory
[59]	Zebra fish	CHD8/idiopathic ASD	Increased head size; reduction in post mitotic enteric neurons
[60]	Zebra fish	FMR1/Fragile X syndrome	Abnormal behaviour; learning memory deficits; impaired craniofacial cartilage development
[61]	Zebra fish	NR3C2/idiopathic ASD	Disruption in sleep and social functions
[62]	Zebra fish	SHANK3/ Phelan-McDermid syndrome	Reduced social nitration and locomotory activity; repetitive swimming behaviour; reduced levels of post synaptic homer1 and presynaptic synaptophysin

ADNP: Activity-dependent neuroprotective protein; ARID1B: AT-rich interaction domain 1B; ASD: Autism spectrum disorder; ASH1L: ASH1-like histone lysine methyltransferase; CHD8: Chromodomain helicase DNA binding protein 8; CNTN5: Contactin-associated protein-like 5; COSMOC: Molybdenum cofactor sulfurase; CYFIP1: Cytoplasmic FMR1 interacting protein; DDX3X: Dead-box helicase 3 X-linked; EHMT1/2: Euchromatic histone lysine methyltransferase 1/2; FMR1: Fragile X messenger ribonucleoprotein 1; FOXP1: Forkhead box protein 1; ES: Embryonic stem; Ih: Hyperpolarization-activated cation; iPSC: Induced pluripotent stem cell; MECP2: Methyl CpG binding protein 2; NMDAR1: N-methyl-D-aspartate receptor 1; NPC: Neural progenitor cell; NR3C2: Nuclear receptor subfamily 3 group c member 2; PTCHD1-AS: Patched domain containing 1-antisense RNA; PTEN: Phosphatase and TENsin homolog; RAB39b: RAS-related protein Rab-39B; SHANK3: SH3- and multiple ankyrin repeats protein 3; TSC1/2: Tuberous sclerosis 1/2; TCF4: Transcription factor 4.

Table 2 Summary of clustered regularly interspaced palindromic repeats-associated protein 9 edited therapeutic targets of autism spectrum disorder

Ref.	In vitro/invivo	Gene mutation/editing method	Observed alterations
[80-82]	BTBR T + tf/J (BTBR), Fmr1 knockout, C57BL/6 mice	mGluR5	Rescued the exaggerated repetitive behaviours in mice caused by fragile X syndrome
[83]	HEK293 cell and Human iPSC (BCRT cell line)	MECP2	Reversal of ASD-associated Rett syndrome-like symptoms
[84]	RX41X iPSC and NOD/SCID female mice	SHANK2	Positive impact on nerve cells was reported like an increase in synapse number, dendritic complexity and length
[85]	C57BL/6 mice, Ube3am-/p+ mice and Ube3am-/pYFP mice on the C57Bl/6	Antisense transcript of UBE3A	Rescued the anatomical and behavioural phenotypes in a mouse model of Angelman syndrome
[86]	HEK293FT cells	FMR1	Fragile X syndrome improved by knocking out the CGG
[89]	Mef2c L35P knock-in mouse	MEF2C	Reversal of autistic-like behaviour

ASD: Autism spectrum disorder; CGG: Cytosine-guanine-guanine; FMR1/Fmr1: Fragile X messenger ribonucleoprotein 1; iPSC: Induced pluripotent stem cell; MECP2: Methyl CpG binding protein 2; MEF2C: Myocyte-specific enhancer factor 2C; mGluR5: Metabotropic glutamate receptor subtype 5; SHANK2: SH3- and multiple ankyrin repeats protein 2.