CRISPR/Cas9 facilitates investigation of neural circuit disease using human iPSCs: mechanism of epilepsy caused by an SCN1A loss-of-function mutation

Abstract

Mutations in SCN1A, the gene encoding the α subunit of Nav1.1 channel, can cause epilepsies with wide ranges of clinical phenotypes, which are associated with the contrasting effects of channel loss-of-function or gain-of-function. In this project, CRISPR/Cas9- and TALEN-mediated genome-editing techniques were applied to induced pluripotent stem cell (iPSC)-based-disease model to explore the mechanism of epilepsy caused by SCN1A loss-of-function mutation. By fluorescently labeling GABAergic subtype in iPSC-derived neurons using CRISPR/Cas9, we for the first time performed electrophysiological studies on SCN1A-expressing neural subtype and monitored the postsynaptic activity of both inhibitory and excitatory types. We found that the mutation c.A5768G, which led to no current of Nav1.1 in exogenously transfected system, influenced the properties of not only Nav current amount, but also Nav activation in Nav1.1-expressing GABAergic neurons. The two alterations in Nav further reduced the amplitudes and enhanced the thresholds of action potential in patient-derived GABAergic neurons, and led to weakened spontaneous inhibitory postsynaptic currents (sIPSCs) in the patient-derived neuronal network. Although the spontaneous excitatory postsynaptic currents (sEPSCs) did not change significantly, when the frequencies of both sIPSCs and sEPSCs were further analyzed, we found the whole postsynaptic activity transferred from the inhibition-dominated state to excitation in patient-derived neuronal networks, suggesting that changes in sIPSCs alone were sufficient to significantly reverse the excitatory level of spontaneous postsynaptic activity. In summary, our findings fill the gap of our knowledge regarding the relationship between SCN1A mutation effect recorded on exogenously transfected cells and on Nav1.1-expressing neurons, and reveal the physiological basis underlying epileptogenesis caused by SCN1A loss-of-function mutation.

Neural Subtype Specification from Human Pluripotent Stem Cells

“…Neurons derived from hPSCs carrying the mutant NRXN1 (Neurexin 1) gene, which is associated with autism and schizophrenia, selectively impaired neurotransmitter release without altering neuronal differentiation or synapse formation (Pak et al, 2015). In GABAergic neurons and glutamatergic neurons that are derived from epilepsy patient iPSCs, the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) but not spontaneous excitatory postsynaptic currents (sEPSC) are significantly lower than those of control and genetically corrected neurons (Liu et al, 2016). These studies indeed suggest defects in transmission in neurons that are produced from patients with mental disorders.…”

Models for discovery of targeted therapy in genetic epileptic encephalopathies

“…With methodological improvements including the use of CRISPR/Cas9 to generate isogenic control cells (Liu et al . ) or specific fluorescent reporters of excitatory and inhibitory neurons (Liu et al . ; Sun et al .…”

“…) or specific fluorescent reporters of excitatory and inhibitory neurons (Liu et al . ; Sun et al . ) the reduced firing of inhibitory neurons became more evident and the accepted view.…”

Muscular dystrophy in a dish: engineered human skeletal muscle mimetics for disease modeling and drug discovery

“…Variability between lines is a significant roadblock to application of patient-based iPSC disease modeling because it might prevent effective comparisons between cell types, limiting the general conclusions that can be drawn for a given condition. This is why most laboratories have embraced the use of CRISPR/Cas9 technology to genetically engineer the desired mutation in an iPSC line from a normal donor [92, 113–116]. This approach creates a matched set of cell lines, with the only difference being the CRISPR-introduced mutation.…”