Page 4 - X. Stem cell biology

[X. Stem cell biology-4]

Safe Scarless Cassette-free Selection of Genome-edited

Human Pluripotent Stem Cells Using Temporary Drug

Resistance

Keun-Tae Kim¹˙#, Ju-Chan Park²˙#, Hyeon-Ki Jang³˙#, Haeseung Lee⁴, Jeongmi Lee⁵, Wankyu Kim⁴,

Sangsu Bae³, Hyuk-Jin Cha²˙*

¹Life Sciences, Sogang University, Seoul 04107, South Korea, ²College of Pharmacy, Seoul National University,

Seoul 08826, South Korea, ³Department of Chemistry, Hanyang University, Seoul 04763, South Korea, ⁴Ewha
Research Center for Systems Biology, Ewha Woman's University, Seoul 03760, South Korea, ⁵School of Pharmacy,
Sungkyunkwan University, Suwon 16419, South Korea

An efficient gene editing technique for use in human pluripotent stem cells (hPSCs) would have great potential
value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However,

the extremely low efficiency of gene editing in hPSCs is a major technical hurdle that remains to be resolved.
Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly

selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155
in hPSCs, which is mediated by selective cellular uptake of the drug, is due to high expression of SLC35F2 in these

cells. Consistent with this, knockout of SLC35F2 with CRISPR-Cas9 or depletion with siRNAs made hPSCs highly
resistant to YM155. Simultaneous gene editing of a gene of interest and transient knockdown of SLC35F2 following

YM155 treatment enabled genome-edited hPSCs to survive because YM155 resistance was temporarily induced,
thereby achieving enriched selection of clonal populations with gene knockout or knock-in. This precise and efficient
genome editing approach took as little as 3 weeks without cell sorting or introduction of additional genes.