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

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.

CRISPR/Cas9-Induced (CTG⋅CAG) n Repeat Instability in the Myotonic Dystrophy Type 1 Locus: Implications for Therapeutic Genome Editing

“…Genome editing with this two-step approach is, however, only realistic for use in cells in vitro and too elaborate for use in vivo. Any direct use of a donor fragment for HDR in muscle cells would make the procedure too highly dependent on correct timing of CRISPR/Cas9 treatment because NHEJ is a much faster process and HDR only occurs in the S and G2-M phases 59, 60, 61. Further sophistication may thus only be possible by genetic or pharmacological suppression of NHEJ62, 63 or by increasing HDR via use of special engineered Cas9 variants, 64 cell-cycle manipulation of the host cell, or precise timing of CRISPR cleavage events.…”

Targeting repair pathways with small molecules increases precise genome editing in pluripotent stem cells

“…Others described an increase of knock-in efficiency in HEK cells 15,17 , while others found no significant effect in rabbit embryos 18 and human stem cells 14,19 . Recently, Greco et al reanalysed the structure and inhibitory properties of SCR7 20 and concluded that SCR7 and its derivates are neither selective nor potent inhibitors of human DNA ligase IV.…”

“…Several studies have tried to increase precise genome editing efficiency by promoting HDR or decreasing NHEJ. Synchronization of cells to the S or G 2 /M phase when homologous recombination occurs increases TNS efficiency in HEK cells (from 26% to 38%), human primary neonatal fibroblast (undetectable to 0.6%) and hESCs (undetectable to 1.6%) 13 and knock-in efficiency in hESCs (from 7 to 41%) 14 . Improved knock-in efficiency was also achieved in HEK cells by suppressing repair proteins like Ku70/80 and DNA ligase IV with siRNA (from 5 to 25%) or by coexpression of adenovirus type 5 proteins 4E1B55K and E4orf6 which mediate degradation of DNA ligase IV among other targets (from 5 to 36%) 15 .…”

Hematopoietic Stem Cell Gene Therapy: Progress and Lessons Learned

“…One such method reported to reduce NHEJ is the inhibition of Ligase IV, which is involved in the final step in the NHEJ pathway (Maruyama et al, 2015; Srivastava et al, 2012; Van Trung Chu et al, 2015). However, other groups have not been able to achieve a significant decrease in NHEJ using this inhibitor (Gutschner et al, 2016; Pinder et al, 2015; Yang et al, 2016). A potential concern with the inhibition of Ligase IV is that decreasing NHEJ levels in the cells may not result in an increase in HDR if the cells have already committed to the end-joining pathway.…”