We then sought to further improve the OPTiKD system by developing an all-in-one targeting approach that would facilitate the rapid and scalable generation of inducible knockdown hPSCs. We constructed a single AAVS1 targeting vector to carry both the inducible shRNA and the CAG-tetR expression cassette (Fig. 3A), and validated this approach by knocking down the expression of an EGFP transgene targeted in the ROSA26 locus (Fig. S3L). Remarkably, this method shared key properties with OPTiKD, such as both the absence of shRNA leakiness (Fig. S3L,M) and rapid, reversible and dose-responsive inducible knockdown (Fig. S3N,O). Thus, this all-in-one strategy, which we named single-step optimized inducible knockdown, or sOPTiKD (Fig. 3A), is as efficient as our original dual targeting approach.
