Enrichment of G2/M cell cycle phase in human pluripotent stem cells enhances HDR-mediated gene repair with customizable endonucleases View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2016-02-18

AUTHORS

Diane Yang, Marissa A Scavuzzo, Jolanta Chmielowiec, Robert Sharp, Aleksandar Bajic, Malgorzata Borowiak

ABSTRACT

Efficient gene editing is essential to fully utilize human pluripotent stem cells (hPSCs) in regenerative medicine. Custom endonuclease-based gene targeting involves two mechanisms of DNA repair: homology directed repair (HDR) and non-homologous end joining (NHEJ). HDR is the preferred mechanism for common applications such knock-in, knock-out or precise mutagenesis, but remains inefficient in hPSCs. Here, we demonstrate that synchronizing synchronizing hPSCs in G2/M with ABT phase increases on-target gene editing, defined as correct targeting cassette integration, 3 to 6 fold. We observed improved efficiency using ZFNs, TALENs, two CRISPR/Cas9, and CRISPR/Cas9 nickase to target five genes in three hPSC lines: three human embryonic stem cell lines, neural progenitors and diabetic iPSCs. neural progenitors and diabetic iPSCs. Reversible synchronization has no effect on pluripotency or differentiation. The increase in on-target gene editing is locus-independent and specific to the cell cycle phase as G2/M phase enriched cells show a 6-fold increase in targeting efficiency compared to cells in G1 phase. Concurrently inhibiting NHEJ with SCR7 does not increase HDR or improve gene targeting efficiency further, indicating that HR is the major DNA repair mechanism after G2/M phase arrest. The approach outlined here makes gene editing in hPSCs a more viable tool for disease modeling, regenerative medicine and cell-based therapies. More... »

PAGES

21264

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/srep21264

DOI

http://dx.doi.org/10.1038/srep21264

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1029521523

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/26887909


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