Two pairs of isogenic N40D MOR human stem cells lines were generated using CRISPR/Cas9 genome editing. Briefly, to convert H1 embryonic stem (ES) cells carrying homozygous AA118 major allele to GG118 homozygous minor alleles, a sgRNA designed from Optimized CRISPR Design Tool (http://crispr.mit.edu/) and Cas9 were expressed using the PX459 vector (Addgene plasmid #62988) and was transfected using the Lipofectamine 3000 reagent (ThermoFisher Scientific, L300015) along with a single stranded oligodeoxynucleotide (ssODN) of 140 base pairs with homology arms flanking the mutation site carrying mutations for G118, a BamHI restriction enzyme site for screening, along with a mutation to mutate the PAM sequence. Individual clones were hand-picked for expansion and screening by PCR and sequencing. Heterozygous clone 9-2 was expanded and transfected for targeting the second allele of OPRM1 Exon 1. The two homozygous G118 knock-in clones were further subcloned before expansion and freezing.
