We aimed to develop optimal conditional loss-of-function platforms using inducible shRNAs or guide RNAs (gRNAs) for CRISPR/Cas9. We reasoned that inserting each element of the TET-ON system into a different genomic safe harbor (GSH; Sadelain et al., 2012) would maximize expression in hPSCs and their differentiated progenies while avoiding potential promoter interference (Shearwin et al., 2005). The AAVS1 and ROSA26 loci appeared particularly suitable for this purpose as these GSHs have been suggested to allow strong expression of various transgenes in hPSCs, including constitutively expressed shRNAs (DeKelver et al., 2010; Hockemeyer et al., 2009; Irion et al., 2007). We first improved the targeting efficiency for both GSHs by developing a CRISPR/Cas9n-based gene-trap strategy to target the human ROSA26 locus (Fig. 1A,B, Fig. S1A) and by refining an existing zinc-finger nuclease (ZFN)-based targeting strategy for the AAVS1 locus (Hockemeyer et al., 2009) (Fig. 1A,B). In both cases, hPSC targeting occurred with very high efficiency (59-100%; Table S1), while neither ROSA26 nor AAVS1 modifications resulted in chromosomal abnormalities (data not shown).
