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The aim is to enhance gene knockout efficiency and optimize sgRNA selection in hPSCs using an inducible Cas9 system.

November 18, 2025Scientific ReportsOpen Access

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

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Optimization improves gene knockout efficiency in hPSCs, indicating better sgRNA selection for gene editing.

The aim is to enhance gene knockout efficiency and optimize sgRNA selection in hPSCs using an inducible Cas9 system.
Developed doxycycline-inducible spCas9-expressing hPSCs (hPSCs-iCas9) line.
Refined parameters for a more effective gene knockout system.
Evaluated gRNA scoring algorithms and used Western blotting to identify ineffective sgRNAs.
Achieved 82-93% INDELs efficiencies for single-gene knockouts and over 80% for double-genes knockouts.
Identified an ineffective sgRNA targeting ACE2 exon 2 that retained protein expression despite high INDEL percentage.
Benchling scored the highest accuracy among tested gRNA scoring algorithms.

Ni et al. (2025) studied this question.

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