Gene Editing of Isogenic iPSC Pairs

Gene editing of isogenic iPSC pairs was performed in collaboration with the Harvard Stem Cell Institute iPSC Core Facility. Briefly, the FA0000011 control iPSC line was characterized for morphology and pluripotency at passage 13 (Supplementary Data 1) and transfected with pCas9GFP (pCas9GFP was a gift from Kiran Musunuru, Addgene plasmid # 44719; http://n2t.net/addgene:44719; RRID: Addgene_44719)^{101 (link)} and independent guide RNAs cloned into pSPgRNA (pSPgRNA was a gift from Charles Gersbach, Addgene plasmid # 47108; http://n2t.net/addgene:47108; RRID: Addgene_47108; see Supplementary Table 2 for list of guide RNAs used)^{102 (link)} using Lipofectamine 3000. GFP-positive cells were sorted and seeded at low density until single cell-derived colonies were formed. Clones were picked and cultured in a 96-well plate format for analysis of target mutations by Sanger DNA sequencing. Clones with a single point mutation in one of the target gene alleles and no modifications to the opposing allele were selected as heterozygous mutants and clones without any detectable modifications in any of the alleles of the target gene were selected as paired controls.