Our study provides the first experimental evidence detailing the functional consequences of the N40D SNP on MOR activation in a human neuronal construct. First, we generated iN cells from human subject-derived stem cells carrying homozygous alleles for N40 MOR or D40 MOR and found that D40 MOR expressing iN cells exhibit stronger inhibitory effects of MOR activation on synaptic release. Second, to validate the functional consequences of the SNP in a system highly controlled for background genetic variation, we used CRISPR/Cas9 mediated gene targeting to: 1) knock-in homozygous D40 alleles into H1ES cells; 2) correct the homozygous D40 alleles in the 03SF iPS cell subject line into N40 alleles, and thus generated two sets of isogenic stem cell lines for highly controlled mechanistic analyses. Third, we demonstrated that the isogenic iN cells not only recapitulated the DAMGO response phenotype of the patient iN cells, but also revealed that the N40D SNP mediates a more robust decrease in excitability and synaptic release, which was accompanied with disruption of a putative N-linked glycosylation motif observed in human D40 MOR 34. The MOR
