Optimization of gene knockout approaches and sgRNA selection in hPSCs with inducible Cas9 expression

Current Cas9-plasmid based systems usually exhibits limited and inconsistent efficiency. Moreover, identifying single-guide RNAs (sgRNAs) with high cleavage efficiency and distinguishing them from ineffective ones remain major challenges in gene knockout experiments. To address these issues, we first generated a doxycycline-inducible spCas9-expressing hPSCs (hPSCs-iCas9) line and then developed it to an optimized gene knockout system through optimizing a set of critical parameters to enhance gene knockout efficiency.