Summary Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore, while mutations in the TARDBP gene (encoding TDP-43) have been associated with ALS, the pathogenic consequences of these mutations remain poorly understood. Using CRISPR/Cas9, we engineered two homozygous knock-in iPSC lines carrying mutations in TARDBP encoding TDP-43 A382T and TDP-43 G348C , two common yet understudied ALS TDP-43 variants. MNs differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization, phosphorylation, solubility, or aggregation compared with isogenic control MNs. However, our results highlight synaptic impairments in both TDP-43 A382T and TDP-43 G348C MN cultures, as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively, our findings argue that MN dysfunction precedes the occurrence of TDP-43 pathology and neurodegeneration in ALS, and further implicates synaptic and excitability defects in the pathobiology of this disease. Highlights MNs differentiated from knock-in iPSCs do not display a neurodegenerative phenotype. Mutant MNs do not show TDP-43 pathology. TDP-43 variants lead to a progressive decline in spontaneous neuronal activity. Functional impairments are accompanied by abnormal synaptic marker expression. eTOC blurb Using CRISPR/Cas9-edited iPSCs, Lépine et al. demonstrate that ALS TDP-43 variants (TDP-43 A382T and TDP43 G348C ) lead to alterations in spontaneous neuronal activity and synaptic abnormalities in the absence of TDP-43 mislocalization, aggregation, or neurodegeneration. These findings imply that TDP-43 pathology is not required to induce MN dysfunction and support the presence of early synaptic impairments prior to MN loss in ALS.