To construct the donor vector, a 2A-eGFP-PGK-Puro cassette (Addgene #31938)39 flanked by two 1kb homology arms PCR amplified from extracted WA25 hESC genomic DNA were cloned into a pUC19 backbone. The two gRNA vectors and the donor vector, as well as a vector expressing Cas9n under the control of CMV promoter (Addgene #41816)40 were transfected into WA25 hESCs with 4D Nucleofector (Lonza) using the P3 Primary Cell 4D-Nucleofector X kit and Program CB-150. After nucleofection, cells were plated in growth medium containing 1× RevitaCell (Thermo Fisher) for improved cell survival rate, and 1µM of Scr7 (Xcessbio) for higher HDR efficiency41. 0.5 µg ml-1 puromycin selection was performed for 10 days starting from 48 hours post-nucleofection. The PGK-Puro sub-cassette flanked by two LoxP sites was removed from the genome after puromycin selection by nucleofection of a Cre recombinase expressing vector (Addgene #13775). Clonal cell lines were established by low-density seeding (1 – 3 cells cm-2) of dissociated single hESCs followed by isolation of hESC colonies after 5-7 days of expansion.