2 Circ Genom Precis Med. 2018; 11: e002327. DOI: 10.1161/CIRCGEN. 118.002327 October 2018 (6 clones of each genotype instead of 3 clones each3). Starting with 1016 cells (homozygous minor, GG), we used CRISPR/Cas9 to introduce a piggyBac transposon harboring a puromycin selection cassette into a TTAA site near rs9349379 (Figure [B]) via homology-directed repair, using previously described techniques. 5 In one targeting, the repair template had G at the site of rs9349379 (preserving the original genotype), whereas in a parallel targeting, the repair template had A (changing the genotype). In either targeting, several clones were identified with homozygous knock-in of the transposon and the rs9349379 allele. After each targeting, each set of clones with the same genotype was pooled and treated with piggyBac transposase as described previously, 5 resulting in scarless removal of the puromycin selection cassettes. We expanded 6 clones of each genotype (GG or AA) and differentiated them in parallel into endothelial cells as described previously, 2 with 7 biological replicates per clone.

We used the same quantitative reverse transcriptase polymerase chain reaction reagents as the previous experiment3 to measure PHACTR1, EDN1, and HGPRT (reference gene) expression. Homozygous major (AA) endothelial cells had significantly higher PHACTR1 expression (22%; P= 1× 10− 7) compared with homozygous minor (GG) endothelial cells, consistent with the A allele conferring enhancer activity for PHACTR1 and with previous rs9349379-PHACTR1 eQTL findings in vascular tissues (Figure [C]). In contrast, we observed no significant difference in EDN1 expression between alternative genotypes, with a trend toward higher expression in AA endothelial cells (rather than significantly lower expression, as observed in the previous experiment3). Thus, although our experiment did not replicate an endothelial rs9349379-EDN1 eQTL, it did confirm previously observed vascular rs9349379-PHACTR1 eQTLs and supported rs9349379 as the causal vari-