Page 3 - X. Stem cell biology

Safe Scarless Cassette-free Selection of Genome-edited Human Pluripotent Stem
Cells Using Temporary Drug Resistance

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Keun-Tae Kim *, Ju-Chan Park *, Hyeon-Ki Jang *, Haeseung Lee , Seokwoo Park , Jumee Kim , Ok-Seon Kwon , Young-Hyun Go , Yan Jin , Wankyu Kim ,
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Jeongmi Lee , Sangsu Bae 4 and Hyuk-Jin Cha 2#
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1Department of Life Sciences, Sogang University, Seoul, Republic of Korea, 2College of Pharmacy, Seoul National University, Seoul, Republic of Korea, 3Department of Chemistry, Hanyang University, Seoul, Republic of
Korea, 4Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, Republic of Korea, 5Ewha Research Center for Systems Biology, Division of Molecular & Life Sciences, Ewha Womans University,
Seoul, Republic of Korea, 6Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea, 7School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Republic of Korea
Abstract
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 substantially 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.
RESULTS

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Figure 1. High expression of SLC35F2 mediates intracellular uptake of YM155 in
hPSCs. (A) A graphical scheme of correlating cell line hPSC scores with cell line drug responses (AUC) Figure 4. YM155 mediated enriched selection in HEK293T. (A) Relative
against each of 543 compounds available in CTRP (B) Correlation of 543 compounds from comparing expression of SLC35F2 in several cells including HEK293T cells through NextBio portal. (B)
Graphical scheme of YM155 mediated enriched selection (YES) approach in HEK293T cells. (C)
AUCs of each compound to hPSC (left) scores and YM155 sensitivity (right). (C) Expression of SLC35F2 in Scheme of CCR5 surrogate vector system. (D) Quantified amount of GFP+ population after
several hESCs from NextBio portal. (D, E) SLC35F2 and POU5F1 gene expression in differentiated cells YES-approach in HEK293T cells. (E) T7E1 assay of samples from (D) (TS: Surrogate vector only,
(hDF: human dermal fibroblast) and hESC (CHA3), hiPSC(SES8) cells. (F) IFC of ɣH2AX in hESCs after C: CCR5 sgRNA only, S: SLC35F2 sgRNA only, C+S: CCR5 and SLC35F2 sgRNA transfected).
YM155 treatment. (G) LC-MS/MS analysis for intracellular YM155 amount between hDF and hESC. (H) (F) Fluorescence microscopic images of from (D). (G) Graph indicates the deep sequencing
analysis targeting CCR5 after YES approach in HEK293T cells. (H) Cell viability of SLC35F2
Graphical scheme of selective uptake of YM155 in hPSCs. reconstituted KO cells after YM155 treatment.
Figure 7. Scarless YES-approach for multiple knockout targets
and knock-in models. (A) T7E1 analysis and (B) indel frequencies with and
without YES-approach in CCR5, HEK2 and HEK3 targets. (C) Fluorescent images of
Cas9-EGFP #1 hESCs (left) and graphical diagram for determining target efficiency
through EGFP reporter system (right). (D) Flow cytometry of EGFP+ and EGFP-
population after YES approach. (E, F) Graphical diagram of knock-in targets at EYA4,
TMEM67 and SLC6A5 loci. (F) Target efficiency of KO and KI of indicative genes. (G)
Plasmid donor DNA-based large insertion targeting AAVS locus with EYFP in hESCs.
B

Figure 2. SLC35F2 enables YM155-induced selective cell death in hPSCs. (A)
Graphical scheme of SLC35F2 target sequence. (B) Experimental scheme of SLC35F2 KO enrichment
(top) and Annexin-V assay after YM155 treatment (bottom). (C) T7E1 assay after YM155 treatment in
SLC35F2 sgRNA transfected hESCs. (D) Sequence information of SLC35F2 KO single clone (KO#1). (E)
Annexin-V assay and (F) IFC for ɣH2AX of KO#1 cells after YM155 treatment. (G) LC-MS/MS analysis for
intracellular YM155 amount in NC and SLC35F2 KO #1 hESCs after YM155 treatment. (H) Phase contrast
image of SLC35F2 KO#5 hESCs after YM155 treatment. (I) SLC35F2 gene expression of KO#1 and KO#5 Figure 5. YM155 based enriched selection of genome-edited hESCs. (A)
hESCs through RT-qPCR (top) and RT-PCR (bottom) (RNA18S for loading control). Graphical scheme of YES-approach with indicative raio of sgRNAs in hESCs. (C: CCR5, S:
SLC35F2 sgRNA). (B) T7E1 assay for CCR5 and SLC35F2 in each clone of hESCs. (C)
Graphical presentation of Indel mutation by indicative conditions (left) and list of read counts
based on deep sequencing (right table). Figure 8. Off-target effect of scarless YES approach Off-target effects
were validated on multiple targets using scarless YES approach. Putative off-target
sites were predicted through off-finder
CONCLUSION

Figure 6. Scarless YES-approach for establishment of CCR5 targeted
hESCs. (A) Graphical scheme of scarless YES-approach. (B) Relative SLC35F2 gene
expression followed by days after siSLC35F2 transfection in hESC. (C) Average ratio of
Figure 3. SLC35F2 KO hPSCs maintain pluripotency and proliferation intact. (A) indel in CCR5 after YM155 YM155 treatment. (D) Phase contrast images of CCR5 targeted
Gene expression of NANOG, SOX2, POU5F1 among hDF, wildtype and SLC35F2 KO#1 hESCs. (B) IFC SLC35F2 expression of CT#1 hESC. (F-H) (F) Pluritpotency related gene and SLC35F2 Contact information
hESC (CT#1) after YM155 treatment. (E) AP staining and pluripotency marker gene and
for LIN28A and SOX2 in KO#1 hESCs. (C) Alkaline phosphatase staining of KO#1 hESCs. (D) Images of
typical 3-germ layers from teratoma from NC and KO#1 hESC. (E) Competition assay between NC and expression of CT#4 and CT#6 hESC, (G) teratoma formation and (H) typical 3-germ layer
KO#1 hESCs. Population of EGFP+ (NC) and EGFP- (KO#1) was quantified through flow cytometry. (F) from CT#6 derived teratoma.
Scatter plot of whole genes from control (NC) and SLC35F2 KO (KO#1) hESCs, (F) Typical pluripotent E-mail: ktkim11@snu.ac.kr
marker genes (POU5F1, SOX2, NANOG, Lin28A) were marked as red circles. (G) Clustering of RNA-seq
samples using t-distributed stochastic neighbor embedding (t-SNE) based on the expression of whole
genes, hPSC signature genes, and cellular transition metal ion homeostasis (GO:0046916) genes. Tel: +82-2-880-7879