First, in the H7 hPSC line (G/G, major/major at rs10889356), we used CRISPR-Cas9 to knock in the minor allele (Figure 4B). We attempted to use a single-strand DNA oligonucleotide as a repair template but were unsuccessful in obtaining any heterozygous or homozygous knock-in clones after screening a large number of clones. We thereafter used a targeting vector with 500-bp homology arms and a puromycin resistance cassette on a transposon that could undergo scarless removal from a TTAA site with piggyBac (Figure 4B). The closest endogenous TTAA site to rs10889356 was 200 bp away. We obtained several clones in which puromycin selection facilitated knock-in of the rs10889356 minor allele into both chromosomes (i.e., homozygous knock-in). We pooled these clones and introduced piggyBac, which yielded a large number of clones in which scarless excision was achieved. Homozygous knock-in (A/A) clones had decreased expression of DOCK7 compared to wild-type clones (down 16%) (Figure 4D). When differentiated into HLCs, homozygous knock-in cells had decreased expression of DOCK7 (down 36%) and substantially increased ANGPTL3 expression (up 60%) (Figure 4D).
