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

• 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?

Transcription Activator-Like Effector Nuclease (TALEN)

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

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

Double safe harbor targeting achieved in human iPSCs

8-week differentiation

AAVS1

CLYBL

Safe harbor targeting achieved in human NSCs

*random integration

Undifferentiated

Differentiated

AAVS1

CLYBL

Global and local gene expression conserved after targeted integration

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

Table S1. Summary of safe-harbor targeted NSCs

Implications for Raphael/Duncan projectProcedure:1. Wash endolymph out of organ of corti and replace with perilymph2. Add furosemide to temporarily block K+ pumps 3. Add sodium caprate to break tight junctions 4. Inject stably expressing reporter stem cells5. Look for cell integration, differentiation, tumors