Figure 2.

Determining the zygosity of the PGP1 line. In each case, a three-primer PCR strategy determined whether the PGP1 line was homozygous or heterozygous at the targeted loci. For the VSX2 locus, primer FW1 is located outside the VSX2 5′HA, RV1 is located inside Cerulean, and RV2 is located outside the VSX2 3′HA. (A) The unedited VSX2 allele (FW1/RV2) should generate a 2.6-kb band, (A′), whereas the edited VSX2 allele (FW1/RV1) should generate a 2.1-kb band. (A′′) A gel image showed bands of 2.6 kb and 2.1 kb for the PGP1 line, whereas the WT hiPSC showed a 2.6-kb band indicating that PGP1 is heterozygous at the VSX2 locus. For the BRN3b locus, FW3 is located outside the BRN3b 5′HA, RV3 is located inside the eGFP, and RV4 is located outside the BRN3b 3′HA. (B) The unedited BRN3b allele (FW3/RV4) should generate a 2.4-kb band, (B′) whereas the edited BRN3b allele (FW3/RV3) should generate a band of 1.7 kb. (B′′) A gel image showed bands of 2.4 kb and 1.7 kb for the PGP1 line, whereas the WT hiPSC showed only a 2.4-kb band, showing that PGP1 is heterozygous at the BRN3b locus. For the RCVRN locus, FW5 is located outside the RCVRN 5′HA, RV5 is located inside mCherry, and RV6 is located outside the RCVRN 3′HA. (C) The unedited RCVRN allele (FW5/RV6) should generate a 2.2-kb band, (C′) whereas the edited RCVRN allele (FW5/RV5) should generate a 1.2-kb band. (C′′) A gel image showed bands of 2.2 kb and 1.2 kb for the PGP1 line, whereas the WT hiPSC showed only a 2.2-kb band, indicating that PGP1 is heterozygous at the RCVRN locus. The original gels that were cropped for clarity in this figure (with white spaces between nonadjacent lanes) can be seen in their entirety in Supplementary Figure S10. Primer information listed in Supplementary Table S8.