1. NIH Center for Regenerative Medicine
Transcription Activator-Like Effector Nuclease (TALEN)-Mediated CLYBL Targeting Enables Enhanced Transgene Expression and One-Step Generation of Dual Reporter Human Induced Pluripotent Stem Cell (iPSC) and Neural Stem Cell (NSC) Lines
Trevor Cerbini, Ray Funahashi, Yongquan Luo, Chengyu Liu, Kyeyoon Park, Mahendra Rao, Nasir Malik, Jizhong Zou
NIH Center for Regenerative Medicine
PLOS One, Jan 2015

2. Ideal safe harbor site (acc. to Papapetrou 2011):
Genome engineering in iPSCs: low transfection efficiency and high rate of apoptosis
Need for system to safely integrate transgenes efficiently – with high transfection efficiency and large number of stable clones
Ideal safe harbor site (acc. to Papapetrou 2011):
>50kb from 5’ end of any gene
>300kb from cancer-related gene and microRNA
Location outside transcriptional units and ultraconserved regions
CLBYL site: stable random integration transgene expression
Question: Can TALEN be used to integrate reporter transgenes efficiently and safely at multiple loci in human iPSCs and NSCs?

3. Transcription Activator-Like Effector Nuclease (TALEN)

4. TALEN can be used to target the safe-harbor CLYBL gene

5. CLYBL safe harbor enables heightened expression of transgenes
5-fold higher HaloTag expression at CLYBL site
10-fold higher Nanoluc expression at CLYBL site
5-fold higher copGFP expression in CLYBL targeted clones

6. Double safe harbor targeting achieved in human iPSCs
AAVS1
CLYBL
8-week differentiation

7. Safe harbor targeting achieved in human NSCs
*random integration
Undifferentiated
AAVS1
Differentiated
CLYBL

8. Global and local gene expression conserved after targeted integration

9. Summary
Transgene integration allowed 10-fold higher expression in CLYBL site than AAVS1 site
38-79% iPSC and 44-90% NSC targeted knock-in of reporter cassettes at either or both safe harbor sites
Simultaneous dual reporter knock in at dual safe harbors in iPSCs and NSCs
Maintained transgene expression and multipotency over long periods of time and after differentiation into cardiomyocytes and neurons
Maintenance of gene expression in loci after transgene integration
Targeted gene-addition efficiencies in human iPSCs

10. Table S1. Summary of safe-harbor targeted NSCs

11. Implications for Raphael/Duncan project
Procedure:
Wash endolymph out of organ of corti and replace with perilymph
Add furosemide to temporarily block K+ pumps
Add sodium caprate to break tight junctions
Inject stably expressing reporter stem cells
Look for cell integration, differentiation, tumors