We generated hESC lines in which a fluorescent reporter gene could be knocked out in an inducible fashion (Fig. 7C). For this, we targeted ROSA26-EGFPd2 reporter hESCs (Fig. S4J,K) with both an inducible EGFP gRNA and a constitutive Cas9 in the AAVS1 locus, each transgene being integrated into one of the two alleles (Fig. 7C,D). This dual targeting approach was rapid (<2 weeks) and efficient (>90% of lines containing both transgenes; Table S1). Remarkably, when individual clonal sublines were grown in the presence of tetracycline we observed decreased EGFPd2 expression in all of the targeted lines, and EGFPd2 homozygous cells showed near-homogeneous loss of at least one copy of the reporter gene as early as 5 days following tetracycline induction (as demonstrated by 50% reduction in EGFPd2 fluorescence, Fig. S8A). Prolonged treatment with tetracycline progressively led to the complete loss of EGFPd2 fluorescence in up to 75% of EGFPd2 homozygous cells (Fig. 7E,F, Fig. S8A,B). Interestingly, co-expression of either two or three copies of the same EGFP gRNA cassette from the same AAVS1 locus was sufficient to significantly increase the
